Electronic detection of biological molecules using thin layers

ABSTRACT

This invention provides novel sensors that facilitate the detection of essentially any analyte. In general, the biosensors of this invention utilize a binding agent (e.g. biomolecule) to specifically bind to one or more target analytes. In preferred embodiments, the biomolecules spans a gap between two electrodes. Binding of the target analyte changes conductivity of the sensor thereby facilitating ready detection of the binding event and thus detection and/or quantitation of the bound analyte. A molecular sensing apparatus comprising.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/970,087, filed onOct. 2, 2001, which claims priority from U.S. Ser. No. 60/292,583, filedon Jun. 11, 2001, which are incorporated herein by reference in theirentirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[Not Applicable]

FIELD OF THE INVENTION

This invention pertains to a biosensor for detecting and/or quantifyinganalytes. More particularly, this invention pertains to a biosensorbased on a detection element that is a single macromolecule spanning twoelectrodes.

BACKGROUND OF THE INVENTION

Biosensors are devices that can detect and/or quantify analytes usingknown interactions between a targeted analyte and a binding agent thatis typically a biological macromolecule such as an enzyme, receptor,nucleic acid, protein, lectin, or antibody. Biosensors have applicationsin virtually all areas of human endeavor. For example, biosensors haveutility in fields as diverse as blood glucose monitoring for diabetics,the recognition of poisonous gas and/or explosives, the detection ofchemicals commonly associated with spoiled or contaminated food, geneticscreening, environmental testing, and the like.

Biosensors are commonly categorized according to two features, namely,the type of macromolecule utilized in the device and the means fordetecting the contact between the binding agent and the targetedanalyte. Major classes of biosensors include enzyme (or catalytic)biosensors, immunosensors and DNA biosensors.

Enzyme (or catalytic) biosensors typically utilize one or more enzymesas the macromolecule and take advantage of the complimentary shape ofthe selected enzyme and the targeted analyte. Enzymes are proteins thatperform most of the catalytic work in biological systems and are knownfor highly specific catalysis. The shape and reactivity of a givenenzyme limits its catalytic activity to a very small number of possiblesubstrates. Enzyme biosensors rely on the specific chemical changesrelated to the enzyme/analyte interaction as the means or recognizingcontact with the targeted analyte. For example, upon interaction with ananalyte, an enzyme biosensor may generate electrons, a coloredchromophore or a change in pH as the result of the relevant enzymaticreaction. Alternatively, upon interaction with an analyte, an enzymebiosensor may cause a change in a fluorescent or chemiluminescent signalthat can be recorded by an appropriate detection system.

Immunosensors utilize antibodies as binding agents. Antibodies areprotein molecules that generally do not perform catalytic reactions, butspecifically bind to particular “target” molecules (antigens).Antibodies are quite specific in their interactions and, unlike mostenzymes, they are capable of recognizing and selectively binding to verylarge bodies such as single cells. Thus, in addition to detection ofsmall analytes, antibody-based biosensors allow for the identificationof certain pathogens such as dangerous bacterial strains.

DNA biosensors typically utilize the complimentary nature of the DNA orRNA. double-strands and are designed for the specific detection ofparticular nucleic acids. A DNA biosensor sensor generally uses asingle-stranded DNA as the binding agent. The nucleic acid material in agiven test sample is placed into contact with the binding agent underconditions where the biosensor DNA and the target nucleic acid analytecan form a hybrid duplex. If a nucleic acid in the test sample iscomplementary to a nucleic acid used in the biosensor, the twointeract/bind. The interaction can be monitored by various means such asa change in mass at the sensor surface or the presence of a fluorescentor radioactive signal. In alternative arrangements, the target nucleicacid(s) are bound to the sensor and contacted with labeled probes toallow for identification of the sequence(s) of interest.

While the potential utility for biosensors is great and while hundredsof biosensors have been described in patents and in the literature,actual commercial use of biosensors remains limited. Aspects ofbiosensors that have limited their commercial acceptance include a lackthe sensitivity and/or speed of detection necessary to accomplishcertain tasks, problems with long term stability, difficultyminiaturizing the sensor, and the like. In addition, a number ofbiosensors must be pre-treated with salts and/or enzyme cofactors, apractice that is inefficient and bothersome.

SUMMARY OF THE INVENTION

This invention pertains to the development of a novel molecular sensingapparatus (biosensor) and to methods of use thereof. In preferredembodiments, the sensing apparatus comprises a first electrode, a secondelectrode, an insulator between the first electrode and the secondelectrode; and a binding agent (e.g. a biological macromolecule)connecting the first electrode to the second electrode. In particularlypreferred embodiments, the binding agent is attached to the electrode ina manner that permits charge to flow from the electrode to the bindingagent or from the binding agent to the electrode. Preferred bindingagents include, but are not limited to, biological macromolecules (e.g.a nucleic acid, a protein, a polysaccharide, a lectin, a lipid, etc.)with a nucleic acid being most preferred. While the nucleic acid can beessentially any length, preferred nucleic acids range in length fromabout 5 nucleotides to about 5,000 nucleotides, more preferably fromabout 8 nucleotides to about 1,000 nucleotides or 500 nucleotides, stillmore preferably from about 10 nucleotides to about 300 nucleotides, andmost preferably from about 15, 20, 25, 30 or 50 nucleotides to about 100nucleotides or 150 nucleotides in length. Typically, the nucleic acid isof sufficient length to specifically hybridize to a target nucleic acidin a complex population of nucleic acids (e.g. total genomic DNA) understringent conditions.

In preferred embodiments, the biological macromolecule is functionalizedwith a chemical group thereby facilitating the attachment of themacromolecule to the electrode(s). Preferred chemical groups include,but are not limited to a sulfate, a sulfhydryl, an amine, an aldehyde, acarboxylic acid, a phosphate, a phosphonate, an alkene, an alkyne, ahydroxyl group, a bromine, an iodine, a chlorine, a light-activatable(labile) group, a group activatable by an electric potential, and thelike. In certain embodiments, the biological macromolecule isfunctionalized with a second biological macromolecule (e.g. a receptor,a receptor ligand, an antibody, an epitope, a nucleic acid, a lectin, asugar, and the like). In preferred embodiments, however, such secondbiological macromolecules exclude nucleic acids.

Preferred insulators are insulators having a resistivity greater thanabout 10′″ ohm-meters, more preferably greater than about 10-2ohm-meters, and most preferably greater than about 10-′, 1, or 10ohm-meters. Suitable insulators include, but are not limited to SiO2,TiO2, ZrO2, quartz, porcelain, ceramic, polystyrene, Teflon (otherhigh-resistivity plastics), an insulating oxide or sulfide of atransition metal in the periodic table of the elements, and the like.

In certain preferred embodiments, the first electrode and the secondelectrode are separated by a distance in the range of 1 to 1010Angstroms. Typically the first electrode and the second electrode areseparated by a distance less than about 300 Angstroms, preferably lessthan about 150 Angstroms, more preferably less than about 70 Angstroms,and most preferably less than about 50 angstroms.

In certain embodiments, the first electrode and/or the second electrodehave a resistivity of less than about 10.2 ohm-meters, preferably lessthan about 10.3 ohm-meters, more preferably less than about 10.4ohm-meters, and most preferably less than about I05, or 10.0 ohm-meters.Particularly preferred electrodes comprise a material such as ruthenium,osmium, cobalt, rhodium, rubidium, lithium, sodium, potassium, vanadium,cesium, beryllium, magnesium, calcium, chromium, molybdenum, silicon,germanium, aluminum, iridium, nickel, palladium, platinum, iron, copper,titanium, tungsten, silver, gold, zinc, cadmium, indium tin oxide,carbon, or a carbon nanotube, certain preferred embodiments, the firstelectrode is functionalized to contain a chemical group that can bederivatized or crosslinked (e.g., a sulfate, a sulfhydryl, an amine, analdehyde, a carboxylic acid, a phosphate, a phosphonate, an alkene, analkyne, a hydroxyl group, a bromine, an iodine, a chlorine, alight-activatable group, a group activatable by an electric potential,etc.). The first and/or second electrode can bear a self-assembledmonolayer (SAM). Particularly preferred SAMs comprise a compoundselected from the group consisting of an alkanethiol, a phospholipid, abola amphiphile, and an oligo(phenylenevinylene).

In a particularly preferred embodiment, the biological macromolecule isattached to the first and/or to the second electrode directly by a thiolgroup or through a linker bearing a thiol group. In another particularlypreferred embodiment, the biological macromolecule is attached to thefirst and/or to the second electrode directly by a phosphonate orthrough a linker bearing a phosphonate. In preferred embodiments, thebiological macromolecule is attached to the first and/or to the secondelectrode by a linker (e.g., DFDNB, DST, ABH, ANB-NOS, EDC, NHS-ASA,SIA, oligo(phenylenevinylene), etc.).

The apparatus can further comprise a substrate (other than the electrodeand/or insulator) where the first electrode and the second electrode areintegrated with the substrate. In certain embodiments, the firstelectrode and the second electrode are integrated with the insulator toform a substrate. The electrodes can be formed in essentially anydesired shape (e.g. convex, concave, textured, corrugated, patterneduniformly, randomly patterned, etc.). Certain preferred electrodeorientations include annular, planar, and orthogonal. In certainembodiments, the first electrode comprises a first surface and a secondelectrode comprises a second surface where the first surface and thesecond surface are not co-planar.

The apparatus can comprise a plurality of electrode pairs. Thus, incertain embodiments, the first electrode and the second electrodecomprise a first electrode pair, and the molecular sensing apparatusfurther comprises a second electrode pair comprising a second firstelectrode and a second electrode. In certain embodiments, the apparatuscomprises at least 3, preferably at least 10 or 20, more preferably atleast 50, 100, or 1,000, and most preferably at least 10,000 or at least1,000,000 electrode pairs.

In certain embodiments, the apparatus further comprises a measurementdevice electrically coupled to the first electrode and to the secondelectrode of at least one said electrode pair. Preferred measurementdevices measure an electromagnetic property selected from the groupconsisting direct electric current, alternating electric current,permitivity, resistivity, electron transfer, electron tunneling,electron hopping, electron transport, electron conductance, voltage,electrical impedance, signal loss, dissipation factor, resistance,capacitance, inductance, magnetic field, electrical potential, chargeand magnetic potential. One particularly preferred measurement device isa potentiostat.

The apparatus can further comprise an electrical circuit electricallycoupled to the first electrode and the second electrode. One suchcircuit comprises an electrical signal gating system (e.g. a CMOS gatingsystem.), and/or a voltage source, and/or a multiplexor, and/or acomputer.

In certain embodiments, the electrodes comprising the first and secondelectrode pairs have attached the same (species of) biologicalmacromolecule. In certain embodiments, different electrode pairs, haveattached different biological molecules.

In certain embodiments, the first electrode and/or the second electrodecomprise a semi-conducting material. Preferred semiconducting materialshave a resistivity ranging from about 10-6 ohm-meters to about 107ohm-meters. Preferred semiconducting materials include, but are notlimited to silicon, dense silicon carbide, boron carbide, Fe3O4,germanium, silicon germanium, silicon carbide, tungsten carbide,titanium carbide, indium phosphide, gallium nitride, gallium phosphide,aluminum phosphide, aluminum arsenide, mercury cadmium telluride,tellurium, selenium, ZnS, ZnO, ZnSe, CdS, ZnTe, GaSe, CdSe, CdTe, GaAs,InP, GaSb, InAs, Tc, PbS, 1nSb, PbTe, PbSe, and tungsten disulfide.

In one embodiment, the apparatus comprises: a first-electrode having afirst surface; a second electrode having a second surface coplanar tothe first surface; an insulator between said first surface and saidsecond surface; and a nucleic acid joining the first electrode to saidsecond electrode.

This invention also provides a method of making a molecular sensingapparatus. In certain embodiments, the method c9rnprises: providing afirst electrode and a second electrode separated by an insulator;contacting the first and the second electrode with a first solutioncomprising a biological macromolecule (e.g., a nucleic acid); placing acharge on the first electrode to attract the biological macromolecule tothe first electrode where the macromolecule attaches to the firstelectrode to form an attached macromolecule; and placing a charge on thesecond electrode to attract a portion of the attached macromolecule tothe second electrode to attach the macromolecule to the secondelectrode. Preferred macromolecules, electrodes, electrodeconfigurations, insulators, measurement devices, circuits, and the like,include, but are not limited to those described above. Where theapparatus comprises multiple electrode pairs, the method can furthercomprise contacting a second electrode pair with a second solutioncomprising a second biological macromolecule; placing a charge on thefirst electrode of the second electrode pair to attract the secondbiological macromolecule to the first electrode of the second electrodepair whereby the second biological macromolecule attaches to said firstelectrode to form an attached second macromolecule; and placing a chargeon the second electrode of said second electrode pair to attract aportion of said attached second macromolecule to attach said secondmacromolecule to said second electrode of said second electrode pair.The first and second solution can be the same or different. Similarly,the first biological macromolecule and the second biologicalmacromolecule can be the same or different.

In still another embodiment, this invention provides a method ofdetecting an analyte. The method involves i) providing molecular sensingapparatus comprising a first electrode and a second electrode separatedby an insulator where said first electrode has a biologicalmacromolecule attached thereto; ii) contacting the attachedmacromolecule with said analyte whereby said analyte binds to saidmacromolecule thereby forming a macromolecule/analyte complex; iii)placing a charge on said second electrode to attract a portion of saidbound analyte to said second electrode where said second analyte isbound to the second electrode such that the macromolecule/analytecomplex forms a connection between the first electrode and the secondelectrode; and iv) detecting the connection between said first and saidsecond electrode. In certain embodiments, the providing comprises:contacting the first electrode with a first solution comprising thebiological macromolecule; and placing a charge on the first electrodewhereby the charge attracts the biological macromolecule to theelectrode and the biological macromolecule attaches to the electrode.Where multiple electrode pairs are present, the method can involverepeating these steps for each electrode pair. The “placing a charge”can, optionally involve placing a charge on the first electrode oppositeto the charge on the second electrode. In certain embodiments, the“detecting” comprises detecting an electromagnetic property selectedfrom the group consisting of direct electric current, alternatingelectric current, permittivity, resistivity, electron transfer, electrontunneling, electron hopping, electron transport, electron conductance,voltage, electrical impedance, signal loss, dissipation factor,resistance, capacitance, inductance, magnetic field, electricalpotential, charge, and magnetic potential. Preferred macromolecules,electrodes, electrode configurations, insulators, measurement devices,circuits, and the like, include, but are not limited to those describedabove.

In still another embodiment, this invention provides a method ofdetecting an analyte, where the method involves: i) providing amolecular sensing apparatus comprising a first electrode and a secondelectrode separated by an insulator where the first electrode has afirst biological macromolecule attached thereto and the second electrodehas a second biological macromolecule attached thereto; ii) contactingthe first attached macromolecule and the second attached macromoleculewith the analyte whereby said analyte binds to the first macromoleculeand to the second macromolecule thereby forming a macromolecule/analytecomplex forming a connection between said first electrode and saidsecond electrode; and iii) detecting the connection between said firstand said second electrode. In certain embodiments, the “providing”comprises contacting the first electrode with a first solutioncomprising the first biological macromolecule; and placing a charge onthe first electrode whereby the charge attracts the first biologicalmacromolecule to the electrode and the biological macromolecule attachesto the electrode. Similarly, in certain embodiments, the “providing”comprises contacting the second electrode with a solution comprising thesecond biological macromolecule; and placing a charge on the secondelectrode whereby the charge attracts the second biologicalmacromolecule to the second electrode and the second biologicalmacromolecule attaches to the second electrode. In certain embodiments,the “detecting” comprises detecting an electromagnetic property selectedfrom the group consisting of direct electric current, alternatingelectric current, permitivity, resistivity, electron transfer, electrontunneling, electron hopping, electron transport, electron conductance,voltage, electrical impedance, signal loss, dissipation factor,resistance, capacitance, inductance, magnetic field, electricalpotential, charge, and magnetic potential. Preferred macromolecules,electrodes, electrode configurations, insulators, measurement devices,circuits, and the like, include, but are not limited to those describedabove.

This invention provides still another method of detecting an analyte.The method involves i) providing a molecular sensing apparatuscomprising a first electrode and a second electrode separated by aninsulator where a biological macromolecule forms a connection betweenthe first electrode and the second electrode; ii) detecting theconnection between the first and the second electrode; iii) contactingthe biological macromolecule (binding agent) with the analyte wherebythe analyte binds to the macromolecule thereby forming amacromolecule/analyte complex; and iv) detecting a difference in theconnection between the first electrode and the second electrode. Incertain embodiments, the “contacting” comprises placing a charge on thefirst and/or the second electrode whereby the charge attracts theanalyte to the biological macromolecule. In certain embodiments, the“providing” comprises contacting the first electrode with a firstsolution comprising the biological macromolecule; and placing a chargeon the first electrode whereby the charge attracts the biologicalmacromolecule to the electrode and the biological macromolecule attachesto the electrode; and placing a charge on the second electrode toattract a portion of the bound macromolecule to the second electrodewhere the macromolecule is bound to the second electrode such that themacromolecule forms a connection between the first electrode and thesecond electrode. In certain embodiments, the “placing a charge”comprises placing a charge on the first electrode opposite to the chargeon the second electrode. The “detecting” can comprise detecting anelectromagnetic property selected from the group consisting of directelectric current, alternating electric current, pennitivity,resistivity, electron transfer, electron tunneling, electron hopping,electron transport, electron conductance, voltage, electrical impedance,signal loss, dissipation factor, resistance, capacitance, inductance,magnetic field, electrical potential, charge and magnetic potential. Inparticularly preferred embodiments, the biological macromolecule isattached to the first electrode by an electrically conductive linker. Incertain embodiments, the binding agent is a nucleic acid and theanalyte, is a protein or a protein complex. Preferred macromolecules,electrodes, electrode configurations, insulators, measurement devices,circuits, and the like, include, but are not limited to those describedabove.

Any of the methods and devices described herein include embodimentswhere the binding agents are not joined to the first electrode and/orthe second electrodes a second or third nucleic acid. Thus, in suchembodiments, where the binding agent is a nucleic acid, a single nucleicacid molecule spans the first and second electrode and linkers orfunctional groups, if present, are not themselves nucleic acids.

Definitions

The term “biosensor” refers to a sensor that uses a biologicalmacromolecule (e.g. nucleic acid, carbohydrate, protein, antibody, etc.)to specifically recognize/bind to a target analyte. The term “molecularsensing apparatus” is used interchangeably with the term “biosensor”.

The term “biological macromolecule” as used herein refers to abiological molecule such as a nucleic acid, protein, antibody,carbohydrate, polysaccharide, lipid, and the like.

The term “electrically conductive” wherein used with reference to alinker, molecule or molecular complex refers to the ability of thatlinker, molecule or molecular complex to pass charge through itselfPreferred electrically conductive molecules have a resistivity lowerthan about 10′ more preferably lower than about 10-4, and mostpreferably lower than about 10-x′ or 10′″ ohm-meters.

The term “electrically coupled” binding agent and an electrode refers toan association between that binding agent and the electrode such thatelectrons can move from the binding agent to the electrode or from theelectrode to the binding agent. Electrical coupling can include directcovalent linkage between the binding agent and the electrode, indirectcovalent coupling (e.g. via a linker), direct or indirect ionic bondingbetween the binding agent and the electrode, or other bonding (e.g.hydrophobic bonding). In addition, no actual bonding may be required andthe binding agent can simply be contacted with the electrode surface.

The term “sensor elem6 nt” as used herein refers to a pair of electrodes(e.g. first electrode 10 and second electrode 12) and associated bindingagent(s) 14 that, when bound by an analyte form a molecular complex thatspans the pair of electrodes.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “nucleic acid” as used herein refers to a deoxyribonucleotideor ribonucleotide in either single- or double-stranded form. The termencompasses nucleic acids, i.e., oligonucleotides, containing knownanalogues of natural nucleotides which have similar or improved bindingproperties, for the purposes desired, as the reference nucleic acid. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones. DNA backbone analogues provided by the invention includephosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene(methylimino), 3′-N-carbamate, morpholino carbamate, andpeptide nucleic acids (PNAs); see Oligonucleotides and Analogues, aPractical Approach, edited by F. Eckstein, IRL Press at OxfordUniversity Press (1991); Antisense Strategies, Annals of the New YorkAcademy of Sciences, Volume 600, Eds. Baserga and Dcnhardt (NYAS 1992);Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research andApplications (1993, CRC Press). PNAs contain non-ionic backbones, suchas N-(2-aminoethyl) glycine units. Phosphorothioate linkages aredescribed in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl.Pharmacol. 144:189-197. Other synthetic backbones encompasses by theterm include methyl-phosphonate linkages or alternatingmethylphosphonate and phosphodiester linkages (Strauss-Soukup (1997)Biochemistry 36: 8692-8698), and benzylphosphonate linkages (Samstag(1996) Antisense Nucleic Acid Drug Dev 6: 153-156). The term nucleicacid is used interchangeably with gene, cDNA, mRNA, oligonucleotideprimer, probe and amplification product.

The term “antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof whichspecifically bind and recognize an analyte (antigen). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, 1 gA, IgD and IgE, respectively. An exemplary immunoglobulin(antibody) structural unit comprises a tetramer. Each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). TheN-4crminus of each chain defines a variable region of about 100 to 110or more amino acids primarily responsible for antigen recognition. Theterms variable light chain (VL) and variable heavy chain (V11) refer tothese light and heavy chains respectively.

Antibodies exist e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide lirkagos in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to V14-C11 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′2 dimer intoan Fab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, Third Edition, W. E. Paul,ed., Raven Press, N.Y. 1993). While various antibody fragments aredefined in terms of the digestion of an intact antibody, one of skillwill appreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies, those synthesized denovo using recombinant DNA methodologies (e.g., single chain Fv), andthose found in display libraries (e.g. phage display libraries).

The phrases “hybridizing specifically to” or “specific hybridization” or“selectively hybridize to”, refer to the binding, duplexing, orhybridizing of a nucleic acid molecule preferentially to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA.

The term “stringent conditions” refers to conditions under which a probewill hybridize preferentially to its target sequence, and to a lesserextent to, or not at all to, other sequences. “Stringent hybridization”and “stringent hybridization wash conditions” in the context of nucleicacid hybridization experiments such as Southern and Northernhybridizations are sequence dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part 1 chapter 2 Overview of principles of hybridization and thestrategy of nucleic. acid probe assays, Elsevier, New York. Generally,highly stringent hybridization and wash conditions are selected to beabout 5° C. lower than the thermal melting point (Tn,) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the targetsequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the T, for a particular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formamidewith 1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72° C. for about 15 minutes. An example of stringent wash conditionsis a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.) suprafor a description of SSC buffer). Often, a high stringency wash ispreceded by a low stringency wash to remove background probe signal. Anexample of a medium stringency wash for a duplex of, e.g., more than 100nucleotides, is 1×SSC at 45° C. for 15 minutes. An example of a lowstringency wash for a duplex of, e.g., more than 100 nucleotides, is4-6×SSC at 40° C. for 15 minutes. In general, a signal to noise ratio of2×(or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids which do not hybridize to each other understringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum. codondegeneracy permitted by the genetic code.

In one particularly preferred embodiment, stringent conditions arecharacterized by hybridization in 1 M NaCl, 10 mM Tris-HCl, pH 8.0,0.01% Triton X-100, 0.1 mg/ml fragmented herring sperm DNA withhybridization at 45° C. with rotation at 50 RPM followed by washingfirst in 0.9 M NaCl, 0.06 M NaH2PO4, 0.0.06 M EDTA, 0.01% Tween-20 at45° C. for 1 hr, followed by 0.075 M NaCl, 0.005 M NaH2PO4, 0.5 mM EDTAat 45° C. for 15 minutes.

A “high resistivity plastic” refers to a plastic with a resistivitygreater than about 10⁻³ ohm-meters, more preferably greater than about10ohm-meters, and most preferably greater than about 10-1, 1, or 10ohm-meters.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a basic biosensoi of this invention. The sensorelement comprises two electrodes 10 and 12 connected by a binding agent(e.g. a biomolecule). Binding of the analyte to the binding agent formsa binding agent/analyte complex spanning the electrodes. The complex iseasily detected using, e.g. electrical means.

FIGS. 2A and 2B illustrate an embodiment of the biosensor comprising twobinding agents, 14 a and 14 b, one on each electrode (FIG. 2B). The twobinding agents are bound by the analyte forming a binding agent/analytecomplex spanning the electrodes. The complex is easily detected using,e.g. electrical means.

FIGS. 3A and 3B illustrate an embodiment of the biosensor comprising abinding agent attached to a first electrode 10 of a pair of electrodes(FIG. 3A). The analyte binds to the binding agent and to the secondelectrode 12 analyte forming a binding agent/analyte complex spanningthe electrodes. The complex is easily detected using, e.g. electricalmeans.

FIGS. 4A and 4B illustrate a simple planar sensor array according tothis invention. FIG. 4A shows a top view, while FIG. 4B illustrates aside view.

FIG. 5 illustrates an aggregation of sensor arrays according to thisinvention.

FIG. 6A through 6C illustrate various sensor embodiments.

FIG. 7 is a schematic a diagram of a support having an array ofelectrode pairs (sensor elements) controlled by a computer.

FIG. 8 is a schematic diagram of a support having an array of electrodepairs (sensor elements).

FIG. 9 is a schematic diagram of a support having an array of electrodepairs and computer system for controlling the energization of eachelectrode pair (sensor element).

FIG. 10 is a schematic diagram of a support having an array of electrodepairs and a computer system with a plurality of voltage sources andmultiplexers for controlling the energization of each electrode pair(sensor element).

FIG. 11 is a diagram of a support having an array of electrode pairs anda computer system with a plurality of switched voltage sources forcontrolling the energization of each electrode pair (sensor element).

FIGS. 12A, 12B, 12C, and 12D illustrate the deposition of alternatingconductor and insulator layers.

FIG. 13 illustrates the use of a biosensor to detect protein/DNAinteractions. A biosensor comprising a nucleic acid 14 is hybridized toa second nucleic acid 24 to form a double-stranded nucleic acid spanningtwo electrodes. Binding of a protein analyte 20 (e.g. DNA bindingprotein) to the nucleic acid changes conductance of the nucleic acidthereby producing a detectable signal.

DETAILED DESCRIPTION

This invention pertains to a novel sensors (biosensors) that are usefulfor detecting a wide range of analytes. The sensors utilize a bindingagent (e.g. a biomolecule) to specifically bind to one or more targetanalytes and thereby confer specificity and selectivity. In preferredembodiments, the binding agent (e.g. biomolecule) spans a gap betweentwo electrodes. Binding of the target analyte changes conductivity, orother electrical properties, of the sensor thereby facilitating readydetection of the binding event and thus detection and/or quantitation ofthe bound analyte. Because the biosensors of this invention provide achange in conductance or charge flow when bound by the target analyte,they are easily read using electronic/electrochemical means and do notrequire the use of detectable labels.

I. Sensor Element Configuration.

One embodiment of a basic biosensor (molecular sensing apparatus) ofthis invention is schematically illustrated in FIG. 1. The sensorcomprises a first electrode 10, a second electrode 12, and a bindingagent (e.g. biomolecule) 14 spanning the gap between the two electrodes.The two electrodes can be separated by an air gap, however, in preferredembodiments, the electrodes are separated by a spacer 16 (e.g. aninsulator, a dielectric, or a semiconductor). The binding agent 14 canbe directly bound to the electrodes or it can be coupled to the firstelectrode 10 and/or the second electrode 12 through one or more linkersor functional groups 18. The binding agent 14 is attached to theelectrodes in a manner that leaves sufficient area of the sensormolecule free to bind with its “cognate” target molecule 20 (the targetanalyte).

In one embodiment, the binding agent 14 is a single-stranded nucleicacid. The nucleic acid is derivatized at each terminus with a linkerthat physically and electrically couples the nucleic acid to therespective electrodes 10 and 12 such that the nucleic acid spans the gapbetween the electrodes. Single-stranded nucleic acids are essentiallynon-conductive. However, when the nucleic acid binding agent iscontacted with a complementary nucleic acid analyte under conditionsthat permit nucleic acid hybridization, the analyte nucleic acid bindsto the sensor nucleic acid via complementary base pairing to form adouble stranded hybrid duplex spanning the electrodes. This doublestranded duplex is electrically conductive. The change in conductivitycaused by such binding is readily detected usingelectrical/electrochemical means.

The binding agent is not limited to a nucleic acid. Any number of otherbinding agents can also be used in such a biosensor. Generally, bindingagents are selected that are capable of specifically binding to aparticular target analyte. Such binding agents include, but are notlimited to proteins, antibodies, lectins, sugars, polysaccharides, andthe like.

While, in preferred embodiments, binding agents are utilized that arenon-conductive by themselves, but form an electrically conductivecomplex when bound to the target analyte. The sensors of this inventionare not limited to such molecules. In certain embodiments it issufficient that the analyte/binding agent complex simply show adifferent conductivity than the binding agent alone.

Alternatively, where the analyte/binding agent complex shows the sameconductivity as the binding agent alone, it is possible to use variouschemical agents that intercalate into the analyte/binding agent complexand change the effective conductivity of that complex. There aretypically intercalation sites, or fewer sites afforded by the bindingagent alone. Thus, the analyte binding complex, by intercalating agreater number of such agents shows a different conductivity.

Intercalating reagents that change the conductivity of a biomolecule ormolecular complex are well known to those of skill in the art. Suchintercalators include, but are not limited to redox-active cations (e.g.Ru(NH₃)₆ ³⁺ and various transition metal/ligand complexes. Transitionmetals are those whose atoms have an incomplete shell of electrons.Suitable transition metals for use in the invention include, but are notlimited to, cadmium (Cd), magnesium (Mg), copper (Cu), cobalt (Co),palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium (Rh),osmium (Os), rhenium (Re), platinum (Pt), scandium (Sc), titanium (Ti),vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni), molybdenum(Mo), technetium (Tc), tungsten (W), and iridium (Ir). That is, thefirst series of transition metal, the platinum metals (Ru, Rh, Pd, Os,Ir and Pt), along with Re, W, Mo and Tc, are preferred. Particularlypreferred are ruthenium, rhenium, osmium, platinum and iron.

The transition metals are complexed with a variety of ligands to formsuitable transition metal complexes, as is well known in the art.Suitable ligands include, but are not limited to, —NH2; pyridine;pyrazine; isonicotinamide; imidazole; bipyridine and substitutedderivative of bipyridine; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline; dipyridophenazine;1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,1,0-phenanthrenequinone diirnine; 1,4,5,8-tetraazaphenanthrene(abbreviated tap); 1,4,8,11-tetraazacyclotetradecane; diaminopyridine(abbreviated damp); porphyrins and substituted derivatives of theporphyrin family.

Such intercalating reagents can also be used to detect mismatchesbetween the binding agent and the target analyte. Thus, for examplewhere the binding agent and the analyte are nucleic acids, intercalatingreagents comprising dimeric naphthyridines will specifically intercalateand localize where there is a G-G mismatch between the binding reagentand the target analyte (see, e.g., Nakatani et al. (2001)Nature/Biotechnology, 19(1): 51-55). Such mismatch specific reagents canbe used t) detect or screen for single nucleotide polymorp_.isms (SNPs).

While FIG. 1 illustrates essentially a single sensor element of thisinvention, various embodiments contemplate the use of a multiplicity ofsensor elements. Thus, in various embodiments, there can exist multiplebinding agents 14 spanning a single pair of electrodes and/or amultiplicity of electrode pairs 20 each electrode pair being spanned byone or more binding agents 14. Because of the small size of the sensorelement, a large number of sensor elements can be placed in a relativelysmall area (e.g. on a chip) thereby increasing sensitivity and improvingsignal to noise (S/N) ratio. In addition, assays can be performed usingsmall quantities of sample. A single substrate/chip can incorporate anumber of different sensor elements facilitatingdetection/quantification of a number of different analytes.

The sensor elements can adopt a wide variety of configurations. Thus,for example, in another embodiment illustrated in FIGS. 2A and 2B, theelectrodes are not spanned by a single binding agent. Rather, a firstbinding agent 14 a is attached to the first electrode 10 and a secondbinding agent 14 b is attached to the second electrode 12 (FIG. 2A).Binding of the analyte 20 to the two binding agents creates anelectrically conductive moiety that spans the gap between the twoelectrodes allowing current to flow between the electrodes and therebyfacilitating detection/quantification of the bound analyte.

Thus, for example, in one embodiment, the first and second bindingagents are each nucleic acids complementary to half of the targetanalyte. When the analyte contacts the binding agents under conditionspermitting hybridization, the two binding agents hybridize to theanalyte forming a double-stranded nucleic acid spanning the twoelectrodes (see, e.g., FIG. 2B).

Still another preferred embodiment is illustrated in FIGS. 3A and 3B. Inthis embodiment, a binding agent 14 is attached to a first electrode 10(FIG. 3A). The target analyte is tagged with a moiety that causes theanalyte to interact with and/or bind to a second electrode. In use, theanalyte 20 binds to, e.g. the second electrode 12 and is bound by thebiological molecule 14. Together the binding agent 14 and the analyte 20bridge the gap between the electrodes resulting in a detectable changein conductance.

In certain embodiments, the analyte is allowed to contact the bindingagent and form a binding agent/analyte complex. Then application of acharge to the second electrode (and, optionally, an opposite charge tothe first electrode) draws the analyte or a portion thereof to thesecond electrode whereby the analyte, or a linker or functional group ofthe analyte and/or the electrode causes the analyte to be linked to thesecond electrode thereby forming the analyte/binding agent complexspanning the two electrodes.

These configurations are simply illustrative of certain preferredembodiments of this invention. Using the teaching provided herein, othersensor element configurations can be readily developed by one ofordinary skill in the art.

While each electrode (electrode pair) can bear a single binding agent14, typically, each electrode (electrode pair) bears a plurality ofbinding agents 14. Thus, in preferred embodiments, each electrode orelectrode pair bears at least two, preferably at least 10, morepreferably at least 50, still more preferably at least 100, and mostpreferably at least 1,000, at least 10,000, at least 100,0000, or atleast 1,000,000 binding agents (e.g., biomolecules) 14.

The electrodes comprising an electrode pair (sensor element) can be ofany convenient dimension. In preferred embodiments, the electrodescomprising an electrode pair are spaced such that the analyte and/or theanalyte/binding agent combination span the gap between the electrodes.In certain embodiments, the electrodes are separated by a distanceranging from about of 1 Angstrom to about 10¹⁰ Angstroms, preferablyfrom about 10 Angstroms to about 10⁵ Angstroms, more preferably fromabout 25 Angstroms to about 10⁴ Angstroms, and most preferably fromabout 40 Angstroms to about 102 Angstroms. Preferred interelectrodespacings are less than about 200 Angstroms, preferably less than about150 Angstroms, more preferably less than about 100 Angstroms, and mostpreferably less than about 50 Angstroms, about 40 Angstroms or about 30Angstroms.

The gap between the electrodes can be an air gap, filled with oxygen orwith an inert gas (e.g. argon, etc.), a vacuum, or the gap can be filledwith an insulator, semiconductor, or a dielectric. In preferredembodiments, the gap between the electrodes is filled with an insulator.Preferred insulators include, elements, compounds or substances thathave a resistivity greater than about 10⁻³ ohm-meters, preferablygreater than about 10⁻² ohm-meters, more preferably greater than about10′″ ohm meters, and most preferably greater than about 10-3 ohm meters.Particularly preferred insulators include, but are not limited to SiO2,TiO2, ZrO2, porcelain, ceramic, glass, clay, polystyrene, Teflon,plastics having a resistivity greater than 10⁻³ ohm-meters, and otherhigh resistivity plastics, insulating oxides or sulfides of thetransition metals in the periodic table of the elements, and the like.

The electrodes are conveniently formed from essentially any conductivematerial. Preferred conductive materials have resistivities of less thanabout 10-′ ohm-meters, preferably less than about 10.4 ohm meters, morepreferably less than about 11-′ ohm meters, and most preferably lessthan about 10-′ ohm meters. In preferred embodiments, the electrodes areformed from materials that include, but are not limited to ruthenium,osmium, cobalt, rhodium, rubidium, lithium, sodium, potassium, vanadium,cesium, beryllium, magnesium, calcium, chromium, molybdenum, silicon,germanium, aluminum, iridium, nickel, palladium, platinum, iron, copper,titanium, tungsten, silver, gold, zinc, cadmium, indium tin oxide,carbon or carbon nanotubes, and alloys or compounds of these materials.

II. Sensor Element Arrays.

Various embodiments of this invention can utilize a single sensorelement. However, in preferred embodiments, a plurality of sensorelements are present, optionally forming an array of sensor elements. Asused herein, an array of sensor elements refers to a plurality of sensorelements aggregated on a common substrate and/or that share one or morecommon electrical connections.

The sensor element arrays can take essentially any conformation that isconvenient to the intended application. Thus, in certain embodiments,the sensor element arrays can comprise planar arrays of sensor elements(see, e.g., FIGS. 4A and 4B) and/or aggregations of such arrays (see,e.g., FIG. 5).

The sensor element arrays are not limited to planar arrays. Virtuallyany configuration can he obtained. Thus, for example, sensor elements orarrays thereof can be placed on one or more walls of a capillary,channel, or microchannel, on one or more walls or floor of a sample well(e.g. in a multi-well plate such as a microtiter plate), on one or moresurfaces of a sensor probe (e.g. an insertable or implantable sensor),and the like. In certain embodiments, the sensor arrays can be stackedto provide three-dimensional arrays.

Certain preferred configurations are illustrated in FIGS. 4 and 6Athrough 6C. Thus, for example, FIG. 4B illustrates a flush-faced sensorarray. The electrodes and insulators are integrated into a multi-layermaterial presenting a flush surface. Analyte(s) or solutions containinganalytes pass across the surface where the analytes are bound by thebinding agent(s) 14. FIG. 6A illustrates an embodiment where theelectrodes protrude from the intervening insulator and thereby form oneor more channels. The channels are useful for guiding reagents/analytes,and the like, e.g. in various microfluidics devices. The bindingagent(s) attached to the electrodes form convenient “detector domains”in such channels. Such devices are readily fabricated by providing amulti-layer material, e.g. as described below, and selectively etchinginsulator away from the electrodes.

Still another embodiment is illustrated in FIG. 6B. In this embodiment,insulator/support is removed between the electrodes thereby formingchannels within the substrate having electrode walls. Optional biasingelectrodes 22 are illustrated in FIG. 6B.

FIG. 6C illustrates a closed channel or well (cross-section) in whichsensor element arrays are present in two walls of the channel.

These configurations are simply illustrative and not intended to belimiting. Using the teaching provided herein, numerous otherconfigurations will be available to one of ordinary skill in the art.

Preferred sensor arrays comprise at least two, preferably at least 10,more preferably at least 100, and most preferably at least 1,000, 10,000or 1,000,000 sensor elements. The sensor elements can all bear the samebiological molecules 14 or various sensor elements can bear differentbiological molecules and show specificity for different analytes. Thus,in certain embodiments, a single sensor array can detect/quantify two ormore, preferably four or more, more preferably 10 or more, still morepreferably 100 or more or 1000 or more, and most preferably 10,000 ormore, 100,000 or more, or even 1,000,000 or more different analytes. Insome molecular sensor apparatus in accordance with the presentinvention, the molecular sensor apparatus comprises 10² to 10¹⁰electrode pairs.

The electrodes comprising the sensor elements of the array(s) can all beseparate, or they can be connected in various combinations. Thus, forexample the first electrodes 10 of all of the sensor elements or for asubset of sensor elements can be electrically connected to form a commonelectrode or “switchably connected to form various electricalconnections as desired. Similarly, additional “biasing” electrodes 22can be connected together or “switchably interconnected.

Numerous methods may be used for addressing the plurality of sensorelements comprising the sensor element arrays of this invention. Severaltechniques are schematically illustrated in FIGS. 7 through 11. Shown inthose figures by way of example are four sensor elements 101, 102, 103,104 and appropriate instrumentation to read them, which typically is avoltammeter incorporating a digital computer.

In FIG. 7, each sensor element (electrode pair) pair 101-104 isindividually addressed by a pair of lines connected to the voltammeter99. By way of example, lines 105, 106 access electrode/counterelectrodepair 101. An appropriate voltage may be applied andconductance/resistance measured by the voltammeter at any given time toany one or more of the pairs of lines connected to the various electrodepairs.

To reduce the number of connections required to address the electrodepairs, alternatives to the direct connection scheme of FIG. 7 areprovided. For example, a row-and column accessing scheme is illustratedin FIG. 8 for electrically energizing some or all of the electrodes. Inthis scheme, one of the electrodes 201, 202 in each column of theplurality of electrode pairs is connected to a common electricalconductor 205 on support 200, and each of the electrodes in each row ofthe plurality of electrode pairs is connected to conductor 207, 208 onthe support 200. Conductors 205, 206 connect to connections C1, C2,respectively, at the edge of support 200 and conductors 207, 208 connectto connections R1, R2, respectively. Each of these connections is thenconnected by a separate line to the voltammeter. As a result, in theconfiguration of FIG. 8, the number of required connections and signallines from the voltammeter has been reduced from 8 to 4.

To enable rapid and sequential energizing/reading of each electrodepair, a computer controlled switching device is beneficial. Theconfiguration of FIG. 9 shows a plurality of first electrodes connectedto a first multiplexer 310. A plurality of second electrodes areconnected to a second multiplexer 320. The first multiplexer is alsoconnected to a first pole of a voltage source/voltammeter 330 thattypically supplies a time varying electrical potential for cyclicvoltammetry described herein. The second multiplexer is also connectedto a second pole of the voltage source/voltammeter. Using addressinglines AO-A3 electrically connected to each of the multiplexers andconnected to latch 340, a computer processor 350 can direct themultiplexers to selectively connect any or all of the first electrodesto the first pole of the voltammeter, and any or all of the secondelectrodes to the second pole of the voltammeter.

As shown in FIG. 10, a plurality of voltage sources are connectedthrough separate sets of multiplexers to each of the electrodes. If afirst electrical potential or range of electrical potentials is requiredat a particular electrode pair, the multiplexers 410, 420 associatedwith the voltage source 430 providing that potential are addressed bythe computer processor 350, typically through a latch 340, therebyconnecting that particular voltage source to the electrode pair inquestion. If a eifferent electrical potential or range of electricalpotentials is required for another electrode pair, the multiplexers 440,450 associated with that different voltage source 460 are addressed bythe computer processor, thereby connecting that voltage source throughthe associated multiplexers 440, 450 to the electrode pair.

If the electrode array in this embodiment has at least a portion of theelectrode pairs independently driveable, as shown in FIG. 8 or FIG. 9,for example, one electrode pair can be driven by one voltagesource/voltammeter while another electrode pair is simultaneously drivenwith another voltage source/voltammeter. Alternatively, the two voltagesources of FIG. 10 can be replaced with a single voltagesource/voltammeter connected to both sets of multiplexers in parallel,allowing two electrode pairs to be driven from the same voltage source.

Instead of a duplicate set of multiplexers for each voltage source asshown in FIG. 10, a plurality of voltage sources/voltammeters 520, 530can be provided as shown in FIG. 11. These voltage sources can beconnected through a computer controlled electrical switch 510 orswitches to a single set of multiplexers 310, 320. As shown in FIG. 11,the computer would direct switch 510 to connect a particular voltagesource/voltammeter to the multiplexers, and would also direct themultiplexers (by signaling their address lines AO-A3) to connect theselected voltage source to the particular electrode pair desired.

Alternatively, the electrical potential applied to each of the electrodepairs in any embodiment can be varied. This is of particular benefitwhen a cassette having a plurality of different sensor elements is used.Such a cassette may require a different range of applied electricalpotential at different sensor elements. Several different embodimentscapable of varying the electrical potential applied to each electrodeare contemplated.

Advantageously, a computer controlled voltage source/voltammeter may beused. A computer controlled voltage source/amperometer is one that canbe addressed by a computer to select a particular electricalpotential/waveform to be supplied. Alternatively it can be programmed tosequentially apply a particular range of electrical potentials over apredetermined time. In such a system, address lines electricallyconnected to the computer and the voltage source allow the computeltoprogram the voltage source to produce the particular electricalpotential to be applied to the electrode pair to be energized.

Additional methods for addressing the plurality of electrode pairs mayalso be used. For example, a plurality of reference electrodes may beplaced in proximity to each of the plurality of electrode pairs in orderto sense the voltage applied thereto. In this way, additional control ofthe voltage waveform may be maintained.

While the foregoing discussion was with reference to voltagesources/amperometers, other means of driving/reading the sensor elementscan be substituted therefor. Such means include, but are not limited toamperometers, coulometers, and the like.

III. Sensor Molecules and Target Analytes.

A) Preferred Sensor Molecules and Target Analytes.

A wide variety of binding agents (binding reagents) 14 can be used inthe devices of this invention and the analytes that can be detectedusing such binding agents are virtually limitless. The binding agentsspecifically bind to at least one analyte (ligand) of interest. Thebinding reagents can be selected from among any molecules known in theart to be capable of, or putatively capable of, specifically binding ananalyte of interest.

Preferred analytes of interest include, but are not limited to a wholecell, a subcellular particle, virus, prion, viroid, nucleic acid,protein, antigen, lipoprotein, lipopolysaccharide, lipid, glycoprotein,carbohydrate moiety, cellulose derivative, antibody or fragment thereof,peptide, hormone, pharmacological agent, cell or cellular components,organic compounds, non-biological polymer, synthetic organic molecule,organo-metallic compounds, or an inorganic molecule present in thesample.

The sample can be derived from, for example, a solid, emulsion,suspension, liquid or gas. Furthermore, the sample may be derived from,for example, body fluids or tissues, water, food, blood, serum, plasma,urine, feces, tissue, saliva, oils, organic solvents, earth, water, air,or food products. The sample may comprise a reducing agent or anoxidizing agent, solubilizer, diluent, preservative, or other suitableagents.

Suitable binding agents (biological molecules) 14 include, but are notlimited to receptors, ligands for receptors, antibodies or bindingportions thereof (e.g., Fab, (Fab)′2), proteins or fragments thereof,nucleic acids, oligonucleotides, glycoproteins, polysaccharides,antigens, epitopes, carbohydrate moieties, enzymes, enzyme substrates,lectins, protein A, protein G, organic compounds, organometalliccompounds, lipids, fatty acids, lipopolysaccharides, peptides, cellularmetabolites, hormones, pharmacological agents, tranquilizers,barbiturates, alkaloids, steroids, vitamins, amino acids, sugars,nonbiological polymers, biotin, avidin, streptavidin, organic linkingcompounds such as polymer resins, lipoproteins, cytokines, lymphokines,hormones, synthetic polymers, organic and inorganic molecules, etc.

It will be apparent from the foregoing that the binding agent (e.g.,biological molecule) 14 and its target analyte 20 can exist as a pair of“binding partners”, e.g. a ligand and its cognate receptor, an antibodyand its epitope, etc. Thus, a biological “binding partner” or a memberof a “binding pair” refers to a molecule or composition thatspecifically binds other molecules to form a binding complex such asantibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid,biotin-avidin, etc.

The term “specifically binds”, as used herein, when referring to abinding agent (e.g., protein, nucleic acid, antibody, etc.), refers to abinding reaction that is determinative of the presence binding agentheterogeneous population of proteins and other biologics. Thus, underdesignated conditions (e.g. immunoassay conditions in the case of anantibody, or stringent hybridization conditions in the case of a nucleicacid), the specified ligand or antibody binds to its particular “target”(e.g. a protein or nucleic acid) and does not bind in a significantamount to other molecules.

The binding partner(s) used in this invention are selected based uponthe targets that are to be identified/quantified. Thus, for example,where the target is a nucleic acid the binding partner is preferably anucleic acid or a nucleic acid binding protein or protein complex (see,e.g, FIG. 13). Where the target is a protein, the binding partner ispreferably a receptor, a ligand, or an antibody that specifically bindsthat protein. Where the target is a sugar or glycoprotein, the bindingpartner is preferably a lectin, and so forth.

B) Preparation of Binding Partners (Capture Agents).

Methods of synthesizing or isolating suitable binding agents are wellknown to those of skill in the art as explained below.

1) Nucleic Acids

Nucleic acids for use as binding agents 14 in this invention can beproduced or isolated according to any of a number of methods well knownto those of skill in the art. In one embodiment, the nucleic acid can bean isolated naturally occurring nucleic acid (e.g., genomic DNA, cDNA,mRNA, etc.). Methods of isolating naturally occurring nucleic acids arewell known to those of skill in the art (see, e.g., Sambrook et al.(1989) Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

In a preferred embodiment, the nucleic acid is created de novo, e.g.through chemical synthesis, e.g., according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers(1981), Tetrahedron Letts., 22(20): 1859-1862, e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al. (1984) NucleicAcids Res., 12: 6159-6168. Purification of oligonucleotides, wherenecessary, is typically performed by either native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson andRegnier (1.983)1 Chrom. 255: 137-149. The sequence of the syntheticoligonucleotides can be verified using the chemical degradation methodof Maxam and Gilbert (1980) in Grossman and Moldave (eds.) AcademicPress, New York, Meth. Enzymol. 65: 499-560.

2) Antibodies/Antibody Fragments.

Antibodies or antibody fragments for use in sensor elements of thisinvention can be produces by a number of methods well known to those ofskill in the art (see, e.g., Harlow & Lane (1988) Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, and Asai (1993)Methods in Cell Biology Vol. 37: Antibodies in Cell Biology, AcademicPress, Inc. N.Y.). In one approach, the antibodies are produced byimmunizing an animal (e.g. a rabbit) with an immunogen containing theepitope it is desired to recognize/capture. A number of immunogens maybe used to produce specifically reactive antibodies. Recombinant proteinis the preferred immunogen for the production of monoclonal orpolyclonal antibodies. Naturally occurring protein may also be usedeither in pure or impure form. Synthetic peptides made as well usingstandard peptide synthesis chemistry (see, e.g., Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield et al. (1.963)1 Am. Chem. Soc., 85: 2149-2156, andStewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. PierceChem. Co., Rockford, Ill.)

Methods of production of polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen is mixed with an adjuvant andanimals are immunized. The animal's immune response to the immunogenpreparation is monitored by taking test bleeds and determining the titerof reactivity to the immunogen. When appropriately high titers ofantibody to the immunogen are obtained, blood is collected from theanimal and antisera are prepared. Further fractionation of the antiserato enrich for antibodies reactive to the immunogen can be done ifdesired. (See Harlow and Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (See, Kohler and Milstein (1976) Eur. J. hnmunol. 6:511-519). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.(1989) Science, 246:1.275-1281.

Antibodies fragments, e.g. single chain antibodies (scFv or others), canalso be produced/selected using phage display technology. The ability toexpress antibody fragments on the surface of viruses that infectbacteria (bacteriophage or phage) makes it possible to isolate a singlebinding antibody fragment from a library of greater than 1010 nonbindingclones. To express antibody fragments on the surface of phage (phagedisplay), an antibody fragment gene is inserted into the gene encoding aphage surface protein (pill) and the antibody fragment-pill fusionprotein is displayed on the phage surface (McCafferty et al. (1990)Nature, 348: 552-554; Hoogenboorn et al. (1991) Nucleic Acids Res. 19:4133-4137).

Since the antibody fragments on the surface of the phage are functional,phage bearing antigen binding antibody fragments can be separated fromnon-binding phage by antigen affinity chromatography (McCafferty et al.(1990). Nature, 348: 552-554). Depending on the affinity of the antibodyfragment, enrichment factors of 20 fold-1,000,000 fold are obtained fora single round of affinity selection. By infecting bacteria with theeluted phage, however, more phage can be grown and subjected to anotherround of selection. In this way, an enrichment of 1000 fold in one roundcan become 1,000,000 fold in two rounds of selection (McCafferty et al.(1990) Nature, 348: 552-554). Thus even when enrichments are low (Markset al. (1991) J. Mol. Biol. 222: 581-597), multiple. rounds of affinityselection can lead to the isolation of rare phage. Since selection ofthe phage antibody library on antigen results in enrichment, themajority of clones bind antigen alter as few as three to four rounds ofselection. Thus only a relatively small number of clones (severalhundred) need to be analyzed for binding to antigen.

Human antibodies can be produced without prior immunization bydisplaying very large and diverse V-gene repertoires on phage (Marks etal. (1991) J. Mol. Biol. 222: 581-597). In one embodiment natural VH andVL repertoires present in human peripheral blood lymphocytes are wereisolated from unimmunized donors by PCR. The V-gene repertoires werespliced together at random using PCR to create a scFv gene repertoirewhich is was cloned into a phage vector to create a library of 30million phage antibodies (Id.). From this single “naive” phage antibodylibrary, binding antibody fragments have been isolated against more than17 different antigens, including haptens, polysaccharides and proteins(Marks et al. (1991) J. Mol. Biol. 222: 581-597; Marks et al. (1993).Bio/Technology. 10: 779-783; Griffiths et al. (1993) EMBO .1.12:725-734; Clackson et al. (1991) Nature. 352: 624-628). Antibodies havebeen produced against self proteins, including human thyroglobulin,immunoglobulin, tumor necrosis factor and CEA (Griffiths et al. (1993)EMBO J. 12: 725-734). It is also possible to isolate antibodies againstcell surface antigens by selecting directly on intact cells. Theantibody fragments are highly specific for the antigen used forselection and have affinities in the I:M to 100 nM range (Marks et al.(1991) J. Mol. Biol. 222: 581-597; Griffiths et al. (1993) EMBO J. 12:725-734). Larger phage antibody libraries result in the isolation ofmore antibodies of higher binding affinity to a greater proportion ofantigens.

3) Binding Proteins.

In one embodiment, the binding partner (capture agent) can be a bindingprotein. Suitable binding proteins include, but are not limited toreceptors (e.g. cell surface receptors), receptor ligands, cytokines,transcription factors and other nucleic acid binding proteins, growthfactors, etc.

The protein can be isolated from natural sources, mutagenized fromisolated proteins or synthesized de novo. Means of isolating naturallyoccurring proteins are well known to those of skill in the art. Suchmethods include but are not limited to well known protein purificationmethods including ammonium sulfate precipitation, affinity columns,column chromatography, gel electrophoresis and the like (see, generally,R. Scopes, (1982) Protein Purification, Springer-Verlag, N.Y.; Dcutscher(1990) Methods in Enzymology Vol. 182: Guide to Protein Purification,Academic Press, Inc. N.Y.).

Where the protein binds a target reversibly, affinity columns bearingthe target can be used to affinity purify the protein. Alternatively theprotein can be recombinantly expressed with a HIS-Tag and purified usingNit+/NTA chromatography.

In another embodiment, the protein can be chemically synthesized usingstandard chemical peptide synthesis techniques. Where the desiredsubsequences are relatively short the molecule may be synthesized as asingle contiguous polypeptide. Where larger molecules are desired,subsequences can be synthesized separately (in one or more units) andthen fused by condensation of the amino terminus of one molecule withthe carboxyl terminus of the other molecule thereby forming a peptidebond. This is typically accomplished using the same chemistry (e.g.,Fmoc, Tboc) used to couple single amino acids in commercial peptidesynthesizers.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield (1962)Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield et al. (1963) 1 Am. Chem. Soc., 85: 2149-2156, andStewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. PierceChem. Co., Rockford, Ill.

In a preferred embodiment, the protein can also be synthesized usingrecombinant DNA methodology. Generally this involves creating a DNAsequence that encodes the binding protein, placing the DNA in anexpression cassette under the control of a particular promoter,expressing the protein in a host, isolating the expressed protein and,if required, renaturing the protein.

DNA encoding binding proteins or subsequences of this invention can beprepared by any suitable method as described above, including, forexample, cloning and restriction of appropriate sequences or directchemical synthesis by methods such as the phosphotriester method ofNarang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester methodof Brown et al. (1979) Meth. Enzyrnol. 68: 109-151; thediethyliphosphoramidite method of Beaucage et al. (1981) Tetra. Lett.,22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066.

The nucleic acid sequences encoding the desired binding protein(s) maybe expressed in a variety of host cells, including E. coli, otherbacterial hosts, yeast, and various higher eukaryotic cells such as theCOS, CHO and HeLa cells lines and myeloma cell lines. The recombinantprotein gene will be operably linked to appropriate expression controlsequences for each host. For E. calf this includes a promoter such asthe T7, trp, or lambda promoters, a ribosome binding site and preferablya transcription termination signal. For eukaryotic cells, the controlsequences will include a promoter and preferably an enhancer derivedfrom immunoglobulin genes, SV40, cytomegalovirus, etc., and apolyadenylation sequence, and may include splice donor and acceptorsequences.

The plasmids can be transferred into the chosen host cell by well-knownmethods such as calcium chloride transformation for E. coli and calciumphosphate treatment or electroporation for mammalian cells. Cellstransformed by the plasmids can be selected by resistance to antibioticsconferred by genes contained on the plasmids, such as the amp, gpt, neoand hvg genes.

Once expressed, the recombinant binding proteins can be purifiedaccording to standard procedures of the art as described above.

4) Sugars and Carbohydrates.

Other binding agents suitable for sensor elements of this inventioninclude, but are not limited to, sugars and carbohydrates. Sugars andcarbohydrates can be isolated from natural sources, enzymaticallysynthesized or chemically synthesized. A route to production of specificoligosaccharide structures is through the use of the enzymes which makethem in vivo; the glycosyltransferases. Such enzymes can be used asregio- and stereoselective catalysts for the in vitro synthesis ofoligosaccharides (Ichikawa et al. (1992) Anal. Biochem. 202: 215-238).Sialyltransferase can be used in combination with additionalglycosyltransferases. For example, one can use a combination ofsialyltransferase and galactosyltransferases. A number of methods ofusing glycosyltransferases to synthesize desired oligosaccharidestructures are known. Exemplary methods are described, for instance, WO96/32491, Ito et al. (1993) Pure Appl. Chem. 65:753, and U.S. Pat. Nos.5,352,670, 5,374,541, and 5,545,553. The enzymes and substrates can becombined in an initial reaction mixture, or alternatively, the enzymesand reagents for a second glycosyltransferase cycle can be added to thereaction medium once the first glycosyltransferase cycle has nearedcompletion. By conducting two glycosyltransferase cycles in sequence ina single vessel, overall yields are improved over procedures in which anintermediate species is isolated.

Methods of chemical synthesis are described by Zhang et al. (1999) 1 Am.Chem. Soc., 121(4): 734-753. Briefly, in this approach, a set ofsugar-based building blocks is created with each block preloaded withdifferent protecting groups. The building blocks are ranked byreactivity of each protecting group. A computer program then determinesexactly which building blocks must be added to the reaction so that thesequences of reactions from fastest to slowest produces the desiredcompound.

IV. Assembling a Sensor.

The biosensors of this invention can be assembled using methods wellknown to those of skill in the art. In general two or more electrodesare provided having an inter-electrode spacing sufficiently small thatthe biomolecule/target analyte complex is capable of carrying chargefrom one electrode to the other. The electrode(s) are then contactedwith the biomolecule(s) 14 in a manner that facilitates the electricalcoupling and physical attachment of the biomolecule(s) to one or bothelectrodes (depending on device configuration). The electrode(s) and/orthe biomolecules can be derivatized so that the molecules selfassemble/attach to the electrode.

A) Providing Two or More Electrodes.

Methods of providing electrodes closely positioned with respect to eachother are well known to those of skill in the art. Thus, for example,electrodes can be precisely positioned using micromanipulators, atomicforce microscope (AFM) or STM tips, and the like. In preferredembodiments, the plurality of electrodes (optional counter electrodes)and the like are typically placed in registered proximity to one anotherby mechanical means, e.g., by using guide posts, alignment pins, guideedges, and the like. Other systems using electrical or magneticregistration means are also available.

In particularly preferred embodiments, the electrodes arefabricated/positioned using micromachining processes (e.g.photolithography) well known in the solid state electronics industry.Commonly, microdevices are constructed from semiconductor materialsubstrates such as crystalline silicon, widely available in the form ofa semiconductor wafer used to produce integrated circuits, or fromglass. Because of the commonality of material(s), fabrication ofmicrodevices from a semiconductor wafer substrate can take advantage ofthe extensive experience in both surface and bulk etching techniquesdeveloped by the semiconductor processing industry for integratedcircuit (IC) production.

Surface etching, used in IC production for defining thin surfacepatterns in a semiconductor wafer, can be modified to allow forsacrificial undercut etching of thin layers of semiconductor materialsto create movable elements. Bulk etching, typically used in ICproduction when deep trenches are formed in a wafer using anisotropicetch processes, can be used to precisely machine edges or trenches inmicrodevices. Both surface and bulk etching of wafers can proceed with“wet processing”, using chemicals such as potassium hydroxide insolution to remove non-masked material from a wafer. For microdeviceconstruction, it is even possible to employ anisotropic wet processingtechniques that rely on differential crystallographic orientations ofmaterials, or the use of electrochemical etch stops, to define variouschannel elements.

Another etch processing technique that allows great microdevice designfreedom is commonly known as “dry etch processing”. This processingtechnique is particularly suitable for anistropic etching of finestructures. Dry etch processing encompasses many gas or plasma phaseetching techniques ranging from highly anisotropic sputtering processesthat bombard a wafer with high energy atoms or ions to displace waferatoms into vapor phase (e.g. ion beam milling), to somewhat isotropiclow energy plasma techniques that direct a plasma stream containingchemically reactive ions against a wafer to induce formation of volatilereaction products.

Intermediate between high energy sputtering techniques and low energyplasma techniques is a particularly useful dry etch process known asreactive ion etching. Reactive ion etching involves directing an ioncontaining plasma stream against a semiconductor, or other, wafer forsimultaneous sputtering and plasma etching. Reactive ion etching retainssome of the advantages of anisotropy associated with sputtering, whilestill providing reactive plasma ions for formation of vapor phasereaction products in response to contacting the reactive plasma ionswith the wafer. In practice, the rate of wafer material removal isgreatly enhanced relative to either sputtering techniques or low energyplasma techniques taken alone. Reactive ion etching therefore has thepotential to be a superior etching process for construction ofmicrodevices, with relatively high anistropic etching rates beingsustainable. The micromachining techniques described above, as well asmany others, are well known to those of skill in the art (see, e.g.,Choudhury (1997) The Handbook of Microlithography, Micromachining, andMicrofabrication, Soc. Photo-Optical Instru. Engineer, Bard & Faulkner(1997) Fundamentals of Microfahricartion). In addition, examples of theuse of micromachining techniques on silicon or borosilicate glass chipscan be found in U.S. Pat. Nos. 5,194,133, 5,132,012, 4,908,112, and4,891,120.

In certain embodiments, the electrodes, particularly electrode arrays ofthis invention are formed as multilayer materials, e.g. alternatinglayers of dialetric and conductor. When etched, cut, or otherwisefractured, the edge of such multilayer materials affords electrodesseparated by dialectric/insulator at extremely high density (closespacing).

Multilayer materials are widely known in the materials community forscientific study and physics applications and their use has beendemonstrated widely (see, e.g., U.S. Pat. Nos. 4,673,623, 4,870,648,4,915,463 and the like).

Such electrode arrays are readily fabricated using sputtering techniques(see, e.g. U.S. Pat. Nos. 5,203,977, 5,486,277, 5,742,471, and thelike). Sputtering is a vacuum coating process where an electricallyisolated cats: ode is mounted in a chamber that can be evacuated andpartially filled with an inert gas. If the cathode material is anelectrical conductor, a direct-current high-voltage power supply is usedto apply the high voltage potential. If the cathode is an electricalinsulator, the polarity of the electrodes is reversed at very highfrequencies to prevent the formation of a positive charge on the cathodethat would stop the ion bombardment process. Since the electrodepolarity is reversed at a radio frequency, this process is referred toas RF-sputtering.

Magnetron sputtering is a more effective form than diode sputtering thatuses a magnetic field to trap electrons in a region near the targetsurface creating a higher probability of ionizing a gas atom. The highdensity of ions created near the target surface causes material to beremoved many times faster than in diode sputtering. The magnetron effectis created by an array of permanent magnets included within the cathodeassembly that produce a magnetic field normal to the electric field.While other sputtering techniques may be used, in particularly preferredembodiments, magnetron sputtering, e.g. as described in U.S. Pat. No.5,486,277, is used to provide the electrode arrays of this invention

B) Attachment of Biomolecules to Electrodes.

The binding agents (e.g. biomolecules) are attached to the electrodesusing methods well known to those of skill in the art. Typically theelectrode(s) and/or the binding agent(s) are derivatized (functionahzed)with reactive moieties (e.g. linkers) that facilitate attachment of theelectrode to the binding agent. Thus, for example in certainembodiments, the binding agent bears a reactive linker (e.g. analiphatic thiol linker) that reacts with the electrode surface or with afunctional group attached thereto, and/or the electrode is derivatizedwith a linker that binds to the bioinolecule.

The linker can be electrically conductive or it can be short enough thatelectrons can pass directly or indirectly between the electrode and thebiological molecule 14.

The manner of linking a wide variety of compounds to various surfaces iswell known and is amply illustrated in the literature. Means of couplingthe biological molecules 14 will be recognized by those of skill in theart. The linkage can be covalent, or by ionic or other non-covalentinteractions. The surface and/or the molecule(s) may be specificallyderivatized to provide convenient linking groups (e.g. sulfur, hydroxyl,amino, etc.).

The linker(s) can be provided as a part of a derivatized binding agentor they can be provided separately. Linkers, when not joined to themolecules to be linked are often either hetero- or homo-bifunctionalmolecules that contain two or more reactive sites that may each form acovalent bond with the respective binding partner (i.e. electrodesurface or biological molecule). When provided as a component thebiological molecule, or attached to the electrode, the linkers arepreferably spacers having one or more reactive sites suitable forbonding to the respective surface or molecule.

Linkers suitable for joining molecules are well known to those of skillin the art and include, but are not limited to any of a variety of, astraight or branched chain carbon linker, or a heterocyclic carbonlinker, amino acid or peptide linkers, and the like. Particularlypreferred linkers include, but are not limited to 4,4′-diphenylethyne,4,4′-diphenylbutadiyne, 4,4′-biphenyl, 1,4-phenylene, 4,47-stilbene,1,4-bicyclooctane, 4,4′-azobenzene, 4,47-benzylideneaniline, and4,4″-terphenyl, oligophenylene vinylenc, and the like (see, e.g., U.S.Pat. No. 6,208,553).

A wide variety of such linkers comprising surface binding groups areknow to those of skill in the art and are often used to produceself-assembling monolayers. Such groups include, but are not limited tothiols (e.g. alkanethiols) (which bind gold and other metals),alkyltrichiorosilane (e.g., which bind silicon/silicon dioxide), alkanecarboxylic acids (e.g., which bind aluminum oxides), derivatives ofethylene glycol, as well as combinations thereof (see, e.g., Ferguson etal. (1993) Macromolecules 26(22): 5870-5875; Prime et al. (1991) Science252:1164-1167; Bain et al. (1989) Angew. Chem. 101: 522-528; Kumar etal. (1994) Langmuir 1C: 1498-1511; Laibinis et al. (1989) Science 245:845-847; Pale-Grosdemange et al. (1991) 1 Am. Chem. Soc., 113: 12-20,and the like). In particularly preferred embodiments, the biologicalmolecules 14 are attached to metal electrodes using thiol linkers (e.g.,alkanethiol linkers).

In certain embodiments, the binding agents are functionalized with achemical group, or a linker bearing a chemical group, that can beactivated by the application of an electrical potential. Such groups arewell known to those of skill in the art and include, but are not limitedto S-benzyloxycarbonyl derivatives, S-benzyl thioethers, S-phenylthioethers, S-4-picolyl thioethers, S-2,2,2-trichloroethoxycarbonylderivatives, S-triphenylmethyl thioethers, and the like. In certainembodiments, the binding agents are functionalized with a chemicalgroup, or a linker bearing a chemical group that can be activated bylight of wavelength ranging from 190 nm to 700 nm. Such chemcial groupsinclude, but are not limited to an aryl azide, a flourinated aryl azide,a benzophenone, and (R,S) (methylene-dioxy)-6-nitrophenyl) ethylcholorformate—(MeNPOC), N-((2-pyridyl, ethyl)-4-azido) salicylamide.

In a particularly preferred embodiment the derivatized biologicalmolecule, in solution, is contacted with the electrode(s). A charge isplaced on the first electrode 10 to attract the biological moleculethereto. Upon contact with the electrode, the derivatized biologicalmolecule binds to the electrode. The derivatized biological molecule canbear two linkers, one for attachment to the first electrode and onederivatized for attachment to the second electrode. In such embodiments,the second linker can be blocked to prevent reaction with the firstelectrode. After the biological molecule has been bound to the firstmolecule the linker is deprotected permitting binding to the secondelectrode.

Thus, for example to span two electrodes with a biological molecule thatis a nucleic acid, the nucleic acid is derivatized with two linkers oneprotected (blocked) thiol and one deprotected (unblocked) thiol. Thefirst electrode 12 is biased positive to attract the nucleic acidthereto whereby the thiol linker binds to the first electrode. The firstelectrode 10 is then biased negative and the second electrode 12 isbiased positive to attract the free end of the nucleic acid to secondelectrode. The blocked thiol linker is deprotected leaving that linkerfree to interact with the second. This results in a nucleic acidspanning gap between the first and the second electrode.

This assembly approach thus uses the device itself, to direct thelocalization and ultimate attachment of the binding agent. Thus, thedevices of this invention are able to electronically self-address eachsensor element with a specific binding agent. The device self-assemblesitself in the sense that no outside process, mechanism, or equipment isneeded to physically direct, position, or place a specific binding agentat a specific location/sensor element/electrode. This self-addressingprocess is both rapid and specific, and can be carried out in either aserial or parallel manner.

The device can be serially addressed with specific binding agent bymaintaining selected sensor element(s)/electrode(s) in a DC mode and atthe opposite charge (potential) to that of a specific binding entity.Other sensor elements/electrodes are maintained at the same charge asthe specific binding agent. In cases where the binding agent is not inexcess of the attachment sites on electrode(s), it is necessary toactivate only one other micro-electrode to affect the electrophoretictransport to the specific micro-location. The specific binding agent israpidly transported (in a few seconds, or preferably less than a second)through the solution, and concentrated directly at the specificelectrode where can covalently bonded to the electrode surface.

The parallel process for addressing sensor elements/electrodes simplyinvolves simultaneously activating a large number (particular group orline) of electrodes so that the same specific binding entity istransported, concentrated, and reacted with more than one specificelectrode.

This approach is simply illustrative. Numerous other approaches can beused to attach the biological molecule to the respective electrode(s).Such approaches include, but are not limited to attachment of chemicalgroups to the surface through the use of photoactivatable chemistries(see, e.g., Sundberg et al. (1995) 1 Am. Chem. Soc.117(49):12050-12057), micro-stamping techniques (see, e.g., Kumar et al.(1994) Langmuir 10(5):1498-1511; Kumar et al. (1993) Appl. Phys. Lett.63(14):2002-2004), and the like.

V. Reading the Sensor.

The sensors of this invention are read using standard methods well knownto those of skill in the art. In particular, the sensors of thisinvention provide a signal that is a change in conductivity(resistivity) of the sensor element(s) as target analytes are bound.

In preferred embodiments, the sensors of this invention are read usingtechniques including, but not limited to amperommetry, voltammetry,capacitance, and impedence. Suitable techniques include, but are notlimited to, electrogravimetry; coulometry (including controlledpotential coulometry and constant current coulometry); voltametry(cyclic voltametry, pulse voltametry (normal pulse voltametry, squarewave voltametry, differential pulse voltametry, Osteryoung square wavevoltametry, and coulostatic pulse techniques); stripping analysis(aniodic stripping analysis, cathiodic stripping analysis, square wavestripping voltammetry); conductance measurements (electrolyticconductance, direct analysis); time-dependent electrochemical analyses(chronoamperometry, chronopotentiometry, cyclic chronopotentiometry andamperometry, AC polography, chronogalvametry, and chronocoulometry); ACimpedance measurement; capacitance measurement; and photoelectrochemistry.

In a preferred embodiment, monitoring electron transfer through thebinding agent/target analyte complex is via amperometric detection. Incertain embodiments, a preferred amperometric detector resembles thenumerous enzyme-based biosensors currently used to monitor bloodglucose, for example. This method of detection involves applying apotential (as compared to a separate reference electrode) between thetwo electrodes comprising a sensor element of this invention. Electrontransfer of differing efficiencies is induced in samples in the presenceor absence of target nucleic add; that is, where the binding agent is anucleic acid, the single stranded binding agent exhibits a differentrate than the probe hybridized to the target sequence. The differingefficiencies of electron transfer result in differing currents beinggenerated in the electrode.

In preferred embodiments, devices for measuring electron transferamperometrically involves sensitive (nanoamp to picoamp) currentdetection and include a means of controlling the voltage potential,usually a potentiostat.

In other preferred embodiments, alternative electron detection modes areutilized. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors can be usedto monitor electron transfer the binding agent/target analyte complex.In addition, other properties of insulators (such as resistance) and ofconductors (such as conductivity, impedance and capacitance) can be usedto monitor electron transfer through the binding agent/target analytecomplex. Finally, any system that generates a current (such as electrontransfer) also generates a small magnetic field, which can be monitoredin some embodiments.

In preferred embodiments, the relatively fast rates of electron transferthrough the binding agent/target analyte complex can facilitate analysisin the frequency (time) domain and thereby dramatically improve signalto noise (S/N) ratios. Thus, in certain embodiments, electron transferis initiated and detected using alternating current (AC) methods. Ingeneral, the use of AC techniques can result in good signals and lowbackground noise. Without being bound by theory, there are a number ofpossible contributors to background noise, or “parasitic” signals, i.e.detectable signals that are inherent to the system but are not theresult of the presence of the target sequence.

However, all of the contributors to parasitic noise generally giverelatively fast signals; that is, the rate of electron transfer throughthe binding agent/target analyte complex is generally significantlyslower than the rate of electron transfer of the parasitic components,such as the contribution of charge carriers in solution, and other“short circuiting” mechanisms. As a result, the parasitic components aregenerally all phase related; that is, they exhibit a constant phasedelay or phase shift that will scale directly with frequency. Thebinding agent/target analyte complex, in contrast, exhibits a time delaybetween the input and output signals, which is independent of frequency.Thus, signal produced by analyte binding will remain relatively constantand relatively large as compared to parasitic background. As aconsequence, at different frequencies, the phase of the system willchange. This is very similar to the time domain detection used influorescent systems.

This difference can be exploited in various methods to decrease thesignal to noise ratio. Accordingly, the preferred detection methodscomprise applying an AC input signal to a binding agent/target analytecomplex. The presence of the binding agent/target analyte complex isdetected via an output signal characteristic of electron transferthrough the binding agent/target analyte complex; that is, the outputsignal is characteristic of the binding agent/target analyte complexrather than the parasitic components or unbound binding agent. Thus, forexample, the output signal will exhibit a time delay dependent on therate of electron transfer through the binding agent/target analytecomplex.

In certain preferred embodiments, the input signals are applied at aplurality of frequencies, since this again allows the distinctionbetween true signal and noise. “Plurality” in this context means atleast two, and preferably more, frequencies. In general, the ACfrequencies will range from about 0.1 Hz to about 10 mHz, with fromabout 1 Hz to 100 KHz being preferred.

In certain preferred embodiments, data analysis is preformed in the timedomain (frequency domain). Thus, for example, cyclic voltammetry isperformed where the signal is analyzed at a harmonic of the fundamentalfrequency. Such measurements can significantly improve the signal tonoise (S/N) ratio.

In preferred embodiments, a cyclic (e.g., sinusoidal sweeping voltage)is applied to the electrode. The response of the binding agent/targetanalyte complex to the sinusoidal voltage is selectively detected at aharmonic of the fundamental frequency of the cyclic voltage rather thanat the fundamental frequency. As a result, a complete frequency spectrumcan be obtained within one cycle.

The step of selectively detecting the voltammetric response comprisesthe step of selectively detecting a current flowing through the bindingagent/target analyte complex at a harmonic of the fundamental frequency.Preferably the harmonic comprises at least one harmonic of the currentabove the fundamental frequency. Typically, the signal is monitored atharmonics at and above the second harmonic of the fundamental frequency.In general, the step of selectively detecting the voltammetric responsecomprises the step of detecting a plurality of higher harmonics of thefundamental frequency within a frequency spectrum of a current flowingthrough the analyte, either through the use of multiple lock-indetectors, or via data acquisition in the time domain, followed by,e.g., Fourier transformation and convolution via computer based methods.Methods of cyclic voltammetry are known to those of skill in the art anddescribe in detail in U.S. Pat. Nos. 6,208,553 and 5,958,215.

VI. Analyte Detection/Quantification.

A) Sample preparation.

Virtually any sample can be analyzed using the devices and methods ofthis invention. Such samples include, but are not limited to body fluidsor tissues, water, food, blood, serum, plasma, urine, feces, tissue,saliva, oils, organic solvents, earth, water, air, or food products. Ina preferred embodiment, the sample is a biological sample. The term“biological sample”, as used herein, refers to a sample obtained from anorganism or from components (e.g., cells) of an organism. The samplemaybe of any biological tissue or fluid. Frequently the sample will be a“clinical sample” which is a sample derived from a patient. Such samplesinclude, but are not limited to, sputum, cerebrospinal fluid, blood,blood fractions (e.g. serum, plasma), blood cells (e.g., white cells),tissue or fine needle biopsy samples, urine, peritoneal fluid, andpleural fluid, or cells therefrom. Biological samples may also includesections of tissues such as frozen sections taken for histologicalpurposes.

Biological samples, (e.g. serum) may be analyzed directly or they may besubject to some preparation prior to use in the assays of thisinvention. Such preparation can include, but is not limited to,suspensipn/dilution of the sample in water or an appropriate buffer orremoval of cellular debris, e.g. by centrifugation, or selection ofparticular fractions of the sample before analysis.

B) Sample Delivery into System.

The sample can be introduced into the devices of this inventionaccording to standard methods well known to those of skill in the art.Thus, for example, the sample can be introduced into the channel throughan injection port such as those used in high pressure liquidchromatography systems. In another embodiment the sample can be appliedto a sample well that communicates to the channel. In still anotherembodiment the sample can be pumped into the channel. Means ofintroducing samples into channels are well known and standard in thecapillary electrophoresis and chromatography arts.

C) Sample Reaction with the Binding Agent.

The analyte containing sample is provided to the sensor element inconditions compatible with or that facilitate binding of the analyte tothe binding agent comprising the sensor element. Thus, for example,where the sensor element is an antibody or protein, reaction conditionsare provided at the sensor element that facilitate antibody binding.Such reaction conditions are well known to those of skill in the art(see, e.g., Techniques for using and manipulating antibodies are foundin Coligan (1991) Current Protocols in Immunology Wiley/Greene, N.Y.;Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and referencescited therein; Goding (1986) Monoclonal Antibodies: Principles andPractice (2d ed.) Academic Press, New York, N.Y.; and Kohler andMilstein (1975) Nature 256: 495-497, and the like).

Similarly, where the binding agent is a nucleic acid the sensor elementis maintained under conditions that facilitate binding of the targetnucleic acid (analyte) to the binding agent comprising the sensorelement(s). Stringency of the reaction can be increased until the sensorshows adequate/desired specificity and selectivity. Conditions suitablefor nucleic acid hybridizations are well known to those of skill in theart (see, e.g., Berger and Kimmel, Guide to MolecularCloninggTechniques, Methods in Enzymology 152 Academic Press, Inc., SanDiego, Calif.; Sambrook et al. (1989) Molecular Cloning—A LaboratoryManual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor Press, NY; Ausubel et al. (1994) Current Protocols in MolecularBiology, Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc.; U.S. Pat. No. 5,017,478;European Patent No. 0,246,864, and the like).

Once the analyte is bound to the binding agent in the sensor element,the sensor is optionally dehydrated and/or stored and/or read.

C) Analyte Detection/Quantitation.

Once introduced into the sensors of this invention, the sample isdetected/quantified using standard methods, e.g. as described above,e.g. amperometry, voltammetry, coulometry, etc. The measurement resultscan be compared to a standard curve, i.e. a series or measurementresults plotted as a function of analyte concentration, which permitsdetermination of analyte concentration. The standard curve can becalculated by/stored in the device performing data acquisition.

V. Cassettes.

In certain embodiments, this invention provides cassettes comprising oneor more sensor elements or sensor element arrays according to thisinvention. In preferred embodiments, cassettes include one or morebiomolecules 14 and/or one or more working electordes 10, 12 and/orbiasing electrodes 22.

Thus, for example in certain embodiments, a cassette with comprise aplurality of biomolecules 14, that are each attached to a pair ofelectrodes. Counter electrodes are optinally provided, e.g. integratedin the layer comprising the working electrodes or provided as acomponent of a second layer comprising the cassette.

In a preferred embodiment, a cassette or apparatus of the inventioncomprises a sample port and/or reservoir and one or more channels forsample delivery onto the sensor element(s) present in the cassette. Themeans for sample delivery can be stationary or movable and can be anyknown in the art, including but not limited to one or more inlets,holes, pores, channels, pipes, microfluidic guides (e.g., capillaries),tubes, and the like.

The channel(s) comprising the cassette of this invention can comprise achannel network, e.g., one or more channels, preferably microchannels.Typically included within a given channel network are channels orreservoirs in which the desired analysis is to take place (analysischannels), and thus the sensor elements of this invention are disposed.Also, optionally included are channels for delivering reagents, buffers,diluents, sample material and the like to the analysis channels.

The cassettes of this invention optionally include separation channelsor matrices separating/fractionating materials transported down thelength of these channels, for analysis, i.e., size or charged basedfractionation of materials, e.g., nucleic acids, proteins etc. Suitableseparation matrices include, e.g., GeneScan.TM. polymers (PerkinElmer-Applied Biosystems Division, Foster City, Calif.). Alternatively,analysis channels are devoid of any separation matrix, and instead,merely provide a channel within which an interaction, reaction etc.,takes place. Examples of microfluidic devices incorporating suchanalysis channels are described in, e.g., PCT Application No. WO98/00231, and U.S. Pat. No. 5,976,336.

Fluids can be moved through the cassette channel system by a variety ofwell known methods, for example: pumps, pipettes, syringes, gravityflow, capillary action, wicking, electrophoresis, electroosmosis,pressure, vacuum, etc. The means for fluid movement may be located onthe cassette or on a separate unit.

The sample can be placed on all of the sensor elements. Alternatively, asample may be placed on particular sensor elements, e.g., by a capillaryfluid transport means. Alternatively, samples may be placed on thesensor element(s) by an automatic pipetter for delivery of fluid samplesdirectly to sensor array, or into a reservoir in a cassette or cassetteholder for later delivery directly to the sensor element(s).

The cassettes of this invention can be fabricated from a wide variety ofmaterials including, but not limited to glass, plastic, ceramic,polymeric materials, elastomeric materials, metals, carbon or carboncontaining materials, alloys, composite foils, silicon and/or layeredmaterials. Supports may have a wide variety of structural, chemicaland/or optical properties. They may be rigid or flexible, flat ordeformed, transparent, translucent, partially or fully reflective oropaque and may have composite properties, regions with differentproperties, and may be a composite of more than one material.

Reagents for conducting assays may be stored on the cassette and/or in aseparate container. Reagents can be stored in a dry and/or wet state. Inone embodiment, dry reagents in the cassette are rehydrated by theaddition of a test sample. In a different embodiment, the reagents arestored in solution in “blister packs” which are burst open due topressure from a movable roller or piston. The cassettes may contain awaste compartment or sponge for the storage of liquid waste aftercompletion of the assay. In one embodiment, the cassette includes adevice for preparation of the biological sample to be tested. Thus, forexample, a filter may be included for removing cells from blood. Inanother example, the cassette may include a device such as a precisioncapillary for the metering of sample.

A cassette or apparatus of the invention can, optionally, comprisereference electrodes, e.g., Ag/AgCI or a saturated calomel electrode(SCE) and/or various biasing/counter-electrodes.

The cassette can also comprise more one layer of electrodes. Thus, forexample, different electrode sets (e.g. arrays of sensor elements) canexist in different lamina of the cassette and thus form a threedimensional array of sensor elements.

VI. Integrated Assay Device/Apparatus.

State-of-the-art chemical analysis systems for use in chemicalproduction, environmental analysis, medical diagnostics and basiclaboratory analysis are preferably capable of complete automation. Suchtotal analysis systems (TAS) (Fillipini et al. (1991) J. Biotechnol. 18:153; Garn et al (1989) Biotechnol. Bioeng. 34: 423; Tshulena (1988)Phys. Scr. T23: 293; Edmonds (1985) Trends Anal. Chem. 4: 220; Stinshoffet al. (1985) Anal. Chem. 57:114R; Guibault (1983) Anal. Chem Symp. Ser.17: 637; Widmer (1983) Trends Anal. Chem. 2: 8) automatically performfunctions ranging from introduction of sample into the system, transportof the sample through the system, sample preparation, separation,purification and detection, including data acquisition and evaluation.

Recently, sample preparation technologies have been successfully reducedto miniaturized formats. Thus, for example, gas chromatography (Widmeret al. (1984) Int. J. Environ. Anal. Chem. 18: 1), high pressure liquidchromatography (Muller et al. (1991) J. High Resolut. Chromatogr. 14:174; Manz et al. (1990) Sensors & Actuators B1:249; Novotny et al., eds.(1985) Microcolumn Separations: Columns, Instrumentation and AncillaryTechniques J. Chromatogr. Library, Vol. 30; Kucera, ed. (1984)Micro-Column High Performance Liquid Chromatography, Elsevier,Amsterdam; Scott, ed. (1984) Small Bore Liquid Chromatography Columns:Their Properties and Uses, Wiley, N.Y.; Jorgenson et al. (1983) 1Chromatogr. 255: 335; Knox et al. (1979), I. Chromatogr. 186:405; Tsudaet al. (1978) Anal. Chem. 50: 632) and capillary electrophoresis (Manzet al. (1992) 1 Chromatogr. 593: 253; Olefirowicz et al. (1990) Anal.Chem. 62:1872; Second Int'l Symp. High—Perf. Capillary Electrophoresis(1990) J. Chromatogr. 516; Ghowsi et al. (1990) Anal. Chem. 62:2714)have been reduced to miniaturized formats.

Similarly, in certain embodiments, this invention provides an integratedassay device (e.g., a TAS) for detecting and/or quantifying one or moreanalytes using the sensor elements, sensor element arrays, or cassettesof this invention.

Thus, in certain embodiments, the cassettes of this invention aredesigned to be inserted into an apparatus, that contains means forreading one or more sensor elements comprising a cassette of thisinvention. The apparatus, optionally includes means for applying one ormore test samples onto the sensor elements or into a receiving port orreservoir and initiating detecting/quantifying one or more analytes.Such apparatus may be derived from conventional apparatus suitablymodified according to the invention to conduct a plurality of assaysbased on a support or cassette. Modifications required include theprovision for, optional, sample and/or cassette handling, multiplesample delivery, multiple electrode reading by a suitable detector, andsignal acquisition and processing means.

Preferred apparatus, in accordance with this invention, thus typicallyinclude instrumentation suitable for performing electrochemicalmeasurements and associated data acquisition and subsequent dataanalysis.

Preferred apparatus also provide means to hold cassettes, optionallyprovide reagents and/or buffers and to provide conditions compatiblewith binding agent/target analyte binding reactions.

A preferred apparatus also comprises an electrode contact means able toelectrically connect the array of separately addressable electrodeconnections of the cassette to an electronic-voltage/waveform generator,e.g., potentiostat. The waveform generator means delivers signalssequentially or simultaneously to independently read a plurality ofsensor elements in the cassette.

The apparatus optionally comprises a digital computer or microprocessorto control the functions of the various components of the apparatus.

The apparatus also comprises signal processing means. In one embodiment,and simply by way of example, the signal processing means comprises adigital computer for transferring, recording, analyzing and/ordisplaying the results of each assay.

The sensor element arrays of this invention are particularly well suitedfor use as detectors in “low sample volume” instrumentation. Suchapplications include, but are not limited to genomic applications suchas monitoring gene expression in plants or animals, parallel geneexpression studies, high throughput screening, clinical diagnosis,single nucleotide polymorphism (SNP) screening, genotyping, and thelike. Certain particularly preferred embodiments, include miniaturizedmolecular assay systems, so-called labs-on-achip, that are capable ofperforming thousands of analyses simultaneously.

Kits.

In certain embodiments, this invention provides kits for practice of themethods and/or assembly of the devices described herein. Preferred kitscomprise a container containing one or more sensor elements according tothe present invention. The sensor elements can be components of a sensorarray and/or can comprise a sensor cassette as describe herein. Incertain embodiments, the kits, optionally, include one or more reagentsand/or buffers for use with the sensors of this invention. The kits canoptionally include materials for sample acquisition, processing, and thelike.

The kits can also include instructional materials containing directions(i.e., protocols) for the practice of the assay methods of thisinvention the use of the cassettes described herein, methods ofassembling sensor elements into various instruments, and the like. Whilethe instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

Sensor Element Formation.

Alternating layers of insulators and conductors are formed by sputteringor vapor deposition (e.g. as described in U.S. Pat. No. 5,414,588. Thelayers consist of a substrate (Alkali-free borosilicate glass (ShaftAF45)), followed by a first conductor, then an insulator, followed by asecond conductor and so forth. The first conductor plus insulator, andthe second conductor plus a second insulator comprise one iteration.Iterations are repeated until the desired number of lamina is achieved.

The position of the conductors and insulators is determined by a mask.Thus, as illustrated in FIG. 12A, conductor 1 has a designated maskand/or mask position of the mask determining the location of itsdeposition. Similarly, the insulator position is determined by the useof a second mask, as illustrated in FIG. 12B, and the position ofconductor 2 is determined by a third mask as illustrated in FIG. 12C.The masks are reused for each subsequent iteration for a total of teniterations.

The sputtering process results in a multi-laminar structure ofalternating conducting and insulating layers where the first conductorlayers are connected to each other and the second layers to be connectedto each other, but not to the first conductor layers (see FIG. 12D)similar to the capacitor described in U.S. Pat. No. 5,414,588.

The conductors are fabricated of gold, and the insulator layers are madeof glass or polystyrene or teflon.

The multilayer structure is cut to expose the thin layers of conductorsand insulators. The exposed surface is then polished smooth. In selectedstructures, the insulator layers are etched further to form a channelbetween the conductive layers.

The first conductor layers are connected to a first macro-electrodeusing common semi-conductor etching methods. The second conductor layersare connected to a second macro-electrode also using commonslni-conductor etching methods.

The macro-electrodes are connected to a voltage source and tested fornon-conductance using an EG&G High Speed Potentiostat/Galvanostat(PerkinElmer Model 283).

Analyte Detection.

The multilayer electrode face is contacted with a capture probe solutioncomprising 30 mu oligonucleotides. The 5 prime end of theoligonucleotides is derivatized with an electrolabile. an alkyl- or arylchloroformate, which can be removed at −1.5 volts in the presence ofLiC1O4/CH₃OH to reveal a thiol group which can then form a covalent bondwith a gold electrode.

The 3 prime end of the oligonucleotide is derivatized with anotherelectrolabile group such as S-henzyloxycarbonyl moiety which can removedat −2.6 volts in DMF and tetrabutyl ammonium chloride. Each of theelectrolabile groups is cleaveable at a unique voltage.

The first conductor is biased with the activation voltage of the 5 primeelectrolabile group on the capture probe thereby exposing the thiolgroup which then attaches to the first conductor.

The second conductor is biases with the activation voltage of the 3prime electrolabile group of the capture probe thereby attaching theprobe to connect to the second conductor. The electrodes are then driedunder nitrogen or argon.

The electrodes are connected to a macro electrodes to a voltage sourceand tested for non-conductance, or a background conductance, is measuredusing a high-speed potentiostat/galvanostat (e.g. Perkin-Elmer, Model283).

The solution comprising the analyte (a nucleic acid comprising asequence complementary to the capture probe) is contacted with thecapture probe and allowed to hybridize to the capture probe on theelectrodes.

The electrodes are dried again under nitrogen or argon. A voltage (4-7volts) is applied again to the electrodes and the current is measured.The measured current of the hybridized nucleic acids is significantlygreater than the current measured for the unhybridized electrodes.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A molecular sensing apparatus comprising: one or more electrodepairs, wherein the electrode pairs comprise: a first electrode; a secondelectrode; and an insulator between said first electrode and said secondelectrode, wherein said insulator comprises a channel between said firstelectrode and said second electrode; and wherein said first electrodeand said second electrode are separated by less than 30 nanometers andsaid apparatus is configured by having parallel electrode portions topermit the formation of a plurality of independent, electrically coupledbinding agent/analyte complexes electrically in parallel between saidfirst electrode and said second electrode.
 2. The molecular sensingapparatus of claim 1, wherein said insulator has a resistivity greaterthan 10⁻³ ohm-meters.
 3. The molecular sensing apparatus of claim 1,wherein said insulator is selected from the group consisting of SiO₂,TiO₂, ZrO₂, quartz, porcelain, ceramic, polystyrene, TEFLON, and aninsulating oxide or sulfide of a transition metal in the periodic tableof the elements.
 4. The apparatus of claim 1, wherein said firstelectrode and said second electrode are separated by a distance lessthan about 70 Angstroms.
 5. The molecular sensing apparatus of claim 1,wherein at least one of said first electrode and said second electrodehas a resistivity of less than 10⁻² ohm-meters.
 6. The molecular sensingapparatus of claim 1, wherein at least one of said first electrode andsaid second electrode has a resistivity of less than 10⁻³ ohm-meters. 7.The molecular sensing apparatus of claim 1, wherein said first electrodeand said second electrode each comprises a material selected from thegroup consisting of ruthenium, osmium, cobalt, rhodium, rubidium,lithium, sodium, potassium, vanadium, cesium, beryllium, magnesium,calcium, chromium, molybdenum, silicon, germanium, aluminum, iridium,nickel, palladium, platinum, iron, copper, titanium, tungsten, silver,gold, zinc, cadmium, indium tin oxide, carbon, and carbon nanotube. 8.The molecular sensing apparatus of claim 1, wherein at least one of saidfirst electrode and said second electrode is functionalized with achemical group that can be derivatized or crosslinked.
 9. The molecularsensing apparatus of claim 8, wherein said chemical group is selectedfrom the group consisting of a sulfate, a sulfhydryl, an amine, analdehyde, a carboxylic acid, a phosphate, a phosphonate, an alkene, analkyne, a hydroxyl, a bromine, an iodine, a chlorine, alight-activatable group, and a group activatable by an electricpotential.
 10. The molecular sensing apparatus of claim 1, wherein atleast one of said first electrode and said second electrode bears aself-assembled monolayer (SAM).
 11. The molecular sensing apparatus ofclaim 10, wherein said SAM comprises a compound selected from the groupconsisting of an alkanethiol, a phospholipid, a bola amphiphile, and anoligo (phenylenevinylene).
 12. The molecular sensing apparatus of claim1, further comprising a substrate that supports the first electrode andthe second electrode, wherein the first electrode and the secondelectrode are integrated with the substrate.
 13. The molecular sensingapparatus of claim 1, wherein the first electrode and the secondelectrode are integrated with the insulator to form a substrate.
 14. Themolecular sensing apparatus of claim 1, wherein said first electrodecomprises a surface with a shape selected from the group consisting ofconvex, concave, textured, corrugated, patterned uniformly, and randomlypatterned.
 15. The molecular sensing apparatus of claim 1, wherein saidfirst electrode and said second electrode are oriented in a formationselected from the group consisting of annular, planar, and orthogonal.16. The molecular sensing apparatus of claim 1, wherein the firstelectrode has a first surface and a said second electrode has a secondsurface, wherein the first surface is not coplanar to the secondsurface.
 17. The apparatus of claim 1, wherein said at least oneelectrode pair comprises a first electrode pair and a second electrodepair.
 18. The molecular sensing apparatus of claim 1, wherein said oneor more electrode pairs are at least 10 electrode pairs.
 19. Themolecular sensing apparatus of claim 1, wherein said one or moreelectrode pairs are at least 1,000 electrode pairs.
 20. The molecularsensing apparatus of claim 1, wherein said one or more electrode pairscomprises about 10² to 10¹⁰ electrode pairs.
 21. The molecular sensingapparatus of claim 1, the molecular sensing apparatus further comprisinga measurement device electrically coupled to each first electrode and toeach second electrode of each electrode pair in said at least oneelectrode pair.
 22. The molecular sensing apparatus of claim 21, whereinsaid measurement device measures an electromagnetic property selectedfrom the group consisting of direct electric current, alternatingelectric current, permitivity, resistivity, electron transfer, electrontunneling, electron hopping, electron transport, electron conductance,voltage, electrical impedance, signal loss, dissipation factor,resistance, capacitance, inductance, magnetic field, electricalpotential, charge and magnetic potential.
 23. The molecular sensingapparatus of claim 1, further comprising an electrical circuitelectrically coupled to the first electrode and the second electrode.24. The molecular sensing apparatus of claim 23, wherein said electricalcircuit comprises an electric signal gating system.
 25. The molecularsensing apparatus of claim 24, wherein said electric signal gatingsystem comprises a CMOS gating system.
 26. The apparatus of claim 18,wherein a first biological macromolecule is attached to the firstelectrode and the second electrode in the first electrode pair, and asecond biological macromolecule is attached to the first electrode andthe second electrode in the second electrode pair; wherein the firstbiological molecule and the second biological molecule are the same. 27.The apparatus of claim 18, wherein a first biological macromolecule isattached to the first electrode and the second electrode in the firstelectrode pair, and a second biological macromolecule is attached to thefirst electrode and the second electrode in the second electrode pair;wherein the first biological molecule and the second biological moleculeare different.
 28. The molecular sensing apparatus of claim 1, furthercomprising a computer electrically coupled to the first electrode andthe second electrode.
 29. The molecular sensing apparatus of claim 1,wherein at least one of the first electrode and the second electrodecomprises a semiconductor material.
 30. The molecular sensing apparatusof claim 29, wherein said semiconductor material has a resistivityranging from 10⁻⁶ Ω-m to 10⁷ Ω-m.
 31. The molecular sensing apparatus ofclaim 29, wherein the semiconductor material is selected from the groupconsisting of silicon, dense silicon carbide, boron carbide, Fe₃O₄,germanium, silicon germanium, silicon carbide, tungsten carbide,titanium carbide, indium phosphide, gallium nitride, gallium phosphide,aluminum phosphide, aluminum arsenide, mercury cadmium telluride,tellurium, selenium, ZnS, ZnO, ZnSc. CdS, ZnTc, GaSc, CdSe, CdTe, GaAs,InP, GaSb, EnAs, Te, PbS, InSb, PbTe, PbSe, and tungsten disulfide. 32.The molecular sensing apparatus of claim 1, wherein a biologicalmacromolecule or macromolecule/analyte complex connects said firstelectrode and said second electrode in said first electrode pair. 33.The molecular sensing apparatus of claim 32, wherein said biologicalmacromolecule is selected from the group consisting of a nucleic acid, aprotein, a polysaccharide, a lectin, and a sugar.
 34. The molecularsending apparatus of claim 32, wherein said biological macromolecule isa deoxyribonucleic acid or a nucleic acid.
 35. The molecular sensingapparatus of claim 32, wherein said biological macromolecule isfunctionalized with a chemical group selected from the group consistingof a sulfate, a sulfhydryl, an amine, an aldehyde, a carboxylic acid, aphosphate, a phosphonate, an alkene, an alkyne, a hydroxyl, a bromine,an iodine, a chlorine, a light-activatable group, and a groupactivatable by an electric potential.
 36. The molecular sensingapparatus of claim 32, wherein the biological macromolecule is attachedto the first electrode by a thiol group.
 37. The molecular sensingapparatus of claim 32, wherein the biological macromolecule is attachedto the first electrode by a phosphorothioate or a phosphonate.
 38. Themolecular sensing apparatus of claim 32, wherein the biologicalmacromolecule is attached to said first electrode by a linker.
 39. Themolecular sensing apparatus of claim 38, wherein said linker is selectedfrom the group consisting of DFDNB, DST, ABH, ANB-NOS, EDC, NHS-ASA, andSIA.
 40. The molecular sensing apparatus of claim 1, wherein a firstbiological macromolecule is attached to said first electrode and asecond biological macromolecule is attached to said second electrode.41. The molecular sensing apparatus of claim 1, wherein said firstelectrode comprises a surface with a shape selected from the groupconsisting of convex, concave, textured, corrugated, patterneduniformly, and randomly patterned.
 42. The molecular sensing apparatusof claim 34, wherein said nucleic acid is deoxyribonucleic acid orribonucleic acid.
 43. The molecular sensing apparatus of claim 1,wherein said first electrode and said second electrode are separated byless than 20 nanometers.
 44. The molecular sensing apparatus of claim43, wherein said first electrode and said second electrode are separatedby less than 15 nanometers.
 45. The molecular sensing apparatus of claim44, wherein said first electrode and said second electrode are separatedby less than 10 nanometers.
 46. A molecular sensing apparatus comprisingone or more electrode pairs in an insulating substrate, wherein a firstelectrode pair in said plurality of electrode pairs comprises a firstelectrode and a second electrode, wherein said first electrode and saidsecond electrode are separated by less than 30 nanometers and saidapparatus is configured by having parallel electrode portions to permitthe formation of a plurality of independent, electrically coupledbinding agent/analyte complexes electrically in parallel between saidfirst electrode and said second electrode.
 47. The molecular sensingapparatus of claim 46, wherein said first electrode and said secondelectrode in said first electrode pair are separated by a distance thatwould allow a biological macromolecule to connect said first electrodeand said second electrode.
 48. The molecular sensing apparatus of claim46, wherein a biological macromolecule connects said first electrode andsaid second electrode.
 49. The molecular sensing apparatus of claim 48,wherein said biological macromolecule is a nucleic acid.
 50. Themolecular sensing apparatus of claim 49, wherein said nucleic acid is adeoxyribonucleic acid or a ribonucleic acid.
 51. The molecular sensingapparatus of claim 47, wherein said distance is in the range of 1Angstrom to 10¹⁰ Angstroms.
 52. The molecular sensing apparatus of claim47, wherein said distance is less than 300 Angstroms.
 53. The molecularsensing apparatus of claim 46, wherein at least one of said firstelectrode and said second electrode has a resistivity of less than 10⁻³ohm-meters.
 54. The molecular sensing apparatus of claim 46, whereinsaid first electrode and said second electrode comprise a materialselected from the group consisting of ruthenium, osmium, cobalt,rhodium, rubidium, lithium, sodium, potassium, vanadium, cesium,beryllium, magnesium, calcium, chromium, molybdenum, silicon, germanium,aluminum, iridium, nickel, palladium, platinum, iron, copper, titanium,tungsten, silver, gold, zinc, cadmium, indium tin oxide, carbon, andcarbon nanotube.
 55. The molecular sensing apparatus of claim 46,wherein at least one of said first electrode and said second electrodeis functionalized with a chemical group that can be derivatized orcrosslinked.
 56. The molecular sensing apparatus of claim 55, whereinsaid chemical group is a sulfate, a sulfhydryl, an amine, an aldehyde, acarboxylic acid, a phosphate, a phosphonate, an alkene, an alkyne, ahydroxyl, a bromine, an iodine, a chlorine, a light-activatable group,or a group activatable by an electric potential.
 57. The molecularsensing apparatus of claim 56, wherein at least one of said firstelectrode and said second electrode is coated with a self-assembledmonolayer.
 58. The molecular sensing apparatus of claim 57, wherein saidself-assembled monolayer comprises a compound selected from the groupconsisting of an alkanethiol, a phospholipid, a bola amphiphile, and anoligo(phenylenevinylene).
 59. The molecular sensing apparatus of claim48, wherein the biological macromolecule is attached to the firstelectrode by a thiol group.
 60. The molecular sensing apparatus of claim48, wherein the biological macromolecule is attached to the firstelectrode by a phosphonate.
 61. The molecular sensing apparatus of claim48, wherein the biological macromolecule is attached to said firstelectrode by a linker.
 62. The molecular sensing apparatus of claim 61,wherein said linker is selected from the group consisting of DFDNB, DST,ABH, ANB-NOS, EDC, NHS-ASA, and SIA.
 63. The molecular sensing apparatusof claim 46, wherein the first electrode has a first surface and thesecond electrode has a second surface, and wherein the first surface isnot coplanar to the second surface.
 64. The molecular sensing apparatusof claim 46, wherein said one or more electrode pairs comprise at leastthree electrode pairs.
 65. The molecular sensing apparatus of claim 46,wherein said one or more electrode pairs comprise at least 10,000electrode pairs.
 66. The molecular sensing apparatus of claim 46,wherein said one or more electrode pairs comprises 10² to 10¹⁰ electrodepairs.
 67. The molecular sensing apparatus of claim 46, the apparatusfurther comprising a measurement device electrically coupled to thefirst electrode and to the second electrode said first electrode pair.68. The molecular sensing apparatus of claim 67, wherein saidmeasurement device measures an electromagnetic property selected fromthe group consisting of direct electric current, alternating electriccurrent, permitivity, resistivity, electron transfer, electrontunneling, electron hopping, electron transport, electron conductance,voltage, electrical impedance, signal loss, dissipation factor,resistance, capacitance, inductance, magnetic field, electricalpotential, charge and magnetic potential.
 69. The molecular sensingapparatus of claim 46, further comprising an electrical circuitelectrically coupled to the first electrode and the second electrode ofsaid first electrode pair.
 70. The molecular sensing apparatus of claim69, wherein said electrical circuit comprises an electric signal gatingsystem.
 71. The molecular sensing apparatus of claim 46, wherein saidbiological molecule connects to said first electrode and said secondelectrode in said first electrode pair.
 72. The molecular sensingapparatus of claim 46, wherein a first biological macromolecule isattached to said first electrode in said first electrode pair, and asecond biological macromolecule is attached to said second electrode insaid first electrode pair.
 73. The molecular sensing apparatus of claim46, further comprising a computer electrically coupled to the firstelectrode and the second electrode of at least one electrode pair insaid one or more electrode pairs.
 74. The molecular sensing apparatus ofclaim 46, wherein at least one of the first electrode and the secondelectrode in an electrode pair in said one or more electrode pairscomprises a semiconductor material.
 75. The molecular sensing apparatusof claim 74, wherein said semiconductor material has a resistivitybetween 10⁻⁶ Ω-m and 10⁻⁷ Ω-m.
 76. The molecular sensing apparatus ofclaim 74, wherein the semiconductor material is selected from the group,consisting of silicon, dense silicon carbide, boron carbide, Fe₃O₄,germanium, silicon germanium, silicon carbide, tungsten carbide,titanium carbide, indium phosphide, gallium nitride, gallium phosphide,aluminum phosphide, aluminum arsenide, mercury cadmium telluride,tellurium, selenium, ZnS, ZnO, ZnSe, CdS, ZnTe, GaSe, CdSe, CdTe, GaAs,InP, GaSb, EnAs, Te, PbS, InSb, PbTe, PbSe, and tungsten disulfide. 77.The molecular sensing apparatus of claim 46, wherein said firstelectrode and said second electrode are separated by less than 20nanometers.
 78. The molecular sensing apparatus of claim 77, whereinsaid first electrode and said second electrode are separated by lessthan 15 nanometers.
 79. The molecular sensing apparatus of claim 78,wherein said first electrode and said second electrode are separated byless than 10 nanometers.